CN116456992A

CN116456992A - Radioactive complexes and radiopharmaceuticals of anti-HER 2 antibodies

Info

Publication number: CN116456992A
Application number: CN202180070240.7A
Authority: CN
Inventors: 河谷稔; 花田贵寿; 殿谷豪太; 武田拓也
Original assignee: Nihon Medi Physics Co Ltd
Current assignee: Nihon Medi Physics Co Ltd
Priority date: 2020-10-16
Filing date: 2021-10-15
Publication date: 2023-07-18

Abstract

The present invention aims to provide a complex which has improved stability as compared with the prior art and which does not impair the efficacy. The complex of the present invention is a complex of an anti-HER 2 antibody site-specifically modified with a peptide and a chelating agent to which a radiometal nuclide is chelated, the peptide and the chelating agent being linked via a linker (L), the linker (L) not containing a thiourea bond.

Description

Radioactive complexes and radiopharmaceuticals of anti-HER 2 antibodies

Technical Field

The present invention relates to radioactive complexes and radiopharmaceuticals of anti-HER 2 antibodies.

Background

HER2 (Human Epidermal Growth Factor Receptor Type: human epidermal growth factor receptor type 2) is a growth factor receptor identified as the gene product of the human oncogene HER2/neu, a transmembrane protein with a molecular weight of about 185 kDa.

As an anti-HER 2 antibody, trastuzumab (Trastuzumab) is known, and is clinically used as an antitumor agent for which breast cancer or gastric cancer is indicated by HER2 overexpression.

Trastuzumab is known as an antibody used in several ADCs (Antibody Drug Conjugate (antibody drug complex)) on the market. ADC is an agent obtained by covalently binding a payload (drug) as a chemotherapeutic agent to an antibody via a linker.

Antibody drugs have high selectivity for targets and few side effects, but on the other hand, the drug efficacy is sometimes insufficient. The chemotherapeutic agent has a strong drug effect, but has a low selectivity to the target, so that the minimum effective amount required to kill cancer cells is high, while the dose cannot be increased too much from the viewpoint of toxicity, so that the maximum tolerance dose is low and the therapeutic dose range is narrow.

According to ADC, a chemotherapeutic agent can be selectively delivered to cancer cells in a large amount, and as a result, an effect is exhibited in a smaller amount, and the chemotherapeutic agent reaching normal cells is reduced, and thus the maximum tolerance dose is also increased, and a widening of the therapeutic dose range is expected.

As one form of ADC (app), there is a radioimmunoconconjugate (radioimmunoconjoate). In radioimmunocomplexes, radionuclides are used in place of payloads.

For example, non-patent documents 1 and 2 describe that ²²⁵ Ac mark Qu TuoThe bead mab is prepared by reacting an isothiocyanate group introduced into DOTA with a terminal amino group of trastuzumab using 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA) as a chelating agent, thereby randomly using ²²⁵ Ac-labeled trastuzumab.

In patent document 1, as an anti-HER 2 antibody, there is described ²²⁵ Ac-labeled trastuzumab ²²⁵ Ac-labeled pertuzumab, wherein, after randomly modifying azido for trastuzumab and pertuzumab, click-reaction with DOTAGA-DBCO labeled with Ac-225, thereby randomly using ²²⁵ Ac-labeled trastuzumab.

In addition, radioimmunocomplexes using gamma-or positron-emitting radionuclides as radionuclides are useful for nuclear medicine examinations. Patent document 2 describes that: complexes of peptides that site-specifically modify the Fc region of antibodies with anti-HER 2 antibodies. Patent document 3 describes that: introducing DTPA into the peptide to modify trastuzumab, and labeling with In-111 to obtain ¹¹¹ In labeling trastuzumab; and introducing DFO into the peptide to modify trastuzumab, labelling with Zr-89 to obtain ⁸⁹ Zr marks trastuzumab.

Prior art literature

Patent literature

Patent document 1: WO2019/125982;

patent document 2: WO2016/186206;

patent document 3: WO2017/217347;

non-patent literature

Non-patent document 1: cancer Res.2003Aug 15;63 (16): 5084-90;

non-patent document 2: clin Cancer Res.2004Jul 1;10 (13): 4489-97.

Disclosure of Invention

Problems to be solved by the invention

However, from the inventors' findings it is clear that: the problem of low stability of the radiolabeled complex of the anti-HER 2 antibody in which a thiourea bond is formed by the reaction of DOTA having an isothiocyanate group introduced thereto and an amino group is that.

In patent document 1, there is no description about site-specific modification of an anti-HER 2 antibody by a peptide. In addition, there is no disclosure or suggestion regarding the problem of the anti-HER 2 antibody in which a thiourea bond is formed.

Means for solving the problems

One embodiment of the invention is a complex of an anti-HER 2 antibody site-specifically modified with a peptide and a chelator to which the radiometal nuclides are chelated, the peptide and chelator being linked via a linker (L) which does not contain a thiourea bond.

Another embodiment of the present invention is a radiopharmaceutical comprising the complex as described above as an active ingredient.

In addition, another embodiment of the present invention is a radiopharmaceutical comprising a complex of a chelator chelated with a radiometal nuclides and an anti-HER 2 antibody as an active ingredient, wherein the complex has a radiochemical purity of 90% or more when stored for 7 days at room temperature, without containing a thiourea bond in the connection between the anti-HER 2 antibody and the chelator.

Unless otherwise indicated, the term "connected" as used herein means directly or indirectly connected.

Effects of the invention

According to the present invention, there is provided a radioactive complex of an anti-HER 2 antibody which has improved stability as compared with the conventional one without impairing the efficacy.

Drawings

[ FIG. 1 ]]Is a graph showing the change in tumor volume over time in tumor-bearing mice of the radioactive complex (example 1), radioactive complex (comparative example 1), antibody control and solvent groups. The vertical axis represents the relative value when the tumor volume at the time of administration of each drug is 1, and the horizontal axis represents the number of days elapsed after administration of each drug. The graph shows the mean ± standard deviation of tumor volumes for each group, "x" indicates the time point at which a significant difference (p < 0.05) was observed relative to the antibody control group, "x" indicates the time point at which a significant difference (p < 0.01) was observed relative to the antibody control group,represents the point in time at which a significant difference (p < 0.05) was observed with respect to the solvent group, < ->Represents the time point at which a significant difference (p < 0.01) was observed with respect to the solvent group.

FIG. 2 is a graph showing changes in body weight of tumor-bearing mice in the group of the radioactive complex (example 1), the group of the radioactive complex (comparative example 1), the antibody control group and the solvent group with time. The vertical axis represents the relative value when the weight at the time of administration of each drug is 1, and the horizontal axis represents the number of days elapsed after administration of each drug. The figures show the mean ± standard deviation of body weight for each group.

FIG. 3 is a graph showing the results of evaluation of antigen binding properties of the radioactive complexes produced in example 1 and comparative example 1. The vertical axis represents a value obtained by dividing the count value in the region of interest (ROI) set on the tumor slice to be used by the area of the ROI, and the horizontal axis represents the cell type of the tumor slice to be used at each evaluation day, after correction by the value obtained by dividing the count value of the standard radiation source by the area of the standard radiation source. The graph shows the mean ± standard deviation of each sample (n=10).

FIG. 4 is a graph showing the results of evaluation of antigen binding properties of the radioactive complexes produced in example 4 and comparative example 4. The vertical axis represents a value obtained by dividing the count value in the region of interest (ROI) set on the tumor slice to be used by the area of the ROI, and the horizontal axis represents the cell type of the tumor slice to be used at each evaluation day, after correction by the value obtained by dividing the count value of the standard radiation source by the area of the standard radiation source. The graph shows the mean ± standard deviation of each sample (n=10).

FIG. 5 is a graph showing the results of evaluation of antigen binding properties of the radioactive complexes produced in example 5 and comparative example 5. The vertical axis represents a value obtained by dividing the count value in the region of interest (ROI) set on the tumor slice to be used by the area of the ROI, and the horizontal axis represents the cell type of the tumor slice to be used at each evaluation day, after correction by the value obtained by dividing the count value of the standard radiation source by the area of the standard radiation source. The graph shows the mean ± standard deviation of each sample (n=10).

FIG. 6 is an image showing a representative example of the results of PET imaging performed on a subcutaneous tumor-bearing model mouse in which SK-OV-3 cells were administered with the radioactive complex produced in example 2. Arrows in the figure indicate tumor-bearing tumors.

FIG. 7 is a graph showing the change in tumor volume with time in tumor-bearing mice in each of the radioactive complex administration groups and each of the ADC agent administration groups. The vertical axis represents the relative value when the tumor volume at the time of administration of each drug is 1, and the horizontal axis represents the number of days elapsed after administration of each drug. The graph shows the mean ± standard deviation of tumor volumes for each group, "×" represents the time point at which a significant difference (p < 0.05) was observed relative to enherrtu (registered trademark) low dose group.

[ FIG. 8 ]]Is a graph showing the change in tumor volume with time in tumor-bearing mice of each of the radioactive complex administration group, the ADC agent administration group, the antibody control group, and the solvent group. The vertical axis represents the relative value when the tumor volume at the time of administration of each drug is 1, and the horizontal axis represents the number of days elapsed after administration of each drug. The graph shows the mean ± standard deviation of tumor volumes for each group, "x" indicates the time point at which a significant difference (p < 0.01) was observed relative to the antibody control group, Represents the time point at which a significant difference (p < 0.01) was observed with respect to the solvent group.

Detailed Description

(1) Radioactive complexes

The present invention is a complex (hereinafter, also referred to as a radioactive complex of the present invention) of an anti-HER 2 antibody site-specifically modified with a peptide and a chelating agent, wherein a radiometal nuclides is chelated to the chelating agent, the peptide and the chelating agent are connected via a linker (L), and the linker (L) does not contain a thiourea bond.

(1-1) radionuclides

The radiometal nuclides contained in the radioactive complex of the present invention are alpha-emitting radionuclides, beta-emitting radionuclides, positron-emitting radionuclides or gamma-emitting radionuclides. In the case of using the radioactive complex of the present invention for the treatment of cancer, it is preferable to use a radionuclide emitting alpha rays or a radionuclide emitting beta rays. In addition, in the case of using the radioactive complex of the present invention for diagnosis or detection of cancer, a positron-emitting radionuclide or a gamma-emitting radionuclide is preferably used. As the radionuclide emitting the α -ray, there may be exemplified: bi-212, bi-213, ac-225, th-227. In addition, as a radionuclide emitting β rays, there may be exemplified: cu-64, Y-90 or Lu-177. In addition, as a positron-emitting radionuclide, there can be exemplified: cu-64, ga-68, Y-86, zr-89. In addition, as a radionuclide emitting γ rays, there may be exemplified: tc-99m or In-111. The radiometal nuclides contained in the radiocomplexes of the invention are more preferably Ac-225, Y-90, lu-177 or Zr-89.

(1-2) antibodies

The antibody contained in the radioactive complex of the present invention is an immunoglobulin that specifically binds to HER2 (hereinafter, also referred to as an antibody used in the present invention). The antibody used in the present invention may be a polyclonal antibody, or a monoclonal antibody, preferably a monoclonal antibody. The source of the antibody is not particularly limited, and examples thereof include: antibodies to non-human animals, antibodies to non-human mammals, and human antibodies, preferably exemplified are: human, rat, mouse and rabbit. In the case where the antibody is derived from a species other than human, it is preferable to perform chimerism or humanization by a well-known technique, and the antibody used in the present invention may be a chimeric antibody, a humanized antibody or a human antibody. In addition, the antibody used in the present invention may be a bispecific antibody.

The antibody used in the radioactive complex of the present invention is more preferably trastuzumab or pertuzumab.

Trastuzumab is a human IgG genetically substituted with the amino acid sequence of the antigen-binding site of mouse monoclonal antibody (4D 5) ₁ Is humanized by gene recombination of a considerable part of the geneMonoclonal antibody consisting of complementarity determining region, human framework region and human IgG of mouse anti-human epithelial growth factor receptor type 2 (HER 2) monoclonal antibody ₁ Is composed of a constant region of (a). Trastuzumab is produced by chinese hamster ovary cells.

In the present specification, trastuzumab is an antibody described in japanese patent publication No. 6-508267, specifically, a humanized antibody comprising the following sequence.

Light chain variable domain amino acid sequence:

[ chemical formula 1]

(SEQ ID NO:1)；

Heavy chain variable domain amino acid sequence:

[ chemical formula 2]

(SEQ ID NO:2)。

Trastuzumab is clinically used as an antitumor agent for indications of breast cancer in which HER2 overexpression is confirmed or gastric cancer in which progress/recurrence of resections cannot be cured in which HER2 overexpression is confirmed, and is available as Herceptin (registered trademark) or various biosimilar (biomimetic pharmaceutical) (trastuzumab BS).

In Herceptin (registered trademark) and its biological analogues, trastuzumab is a glycoprotein (molecular weight: about 148,000) composed of 2 molecules of an H chain (γ1 chain) containing 450 amino acid residues and 2 molecules of an L chain (κchain) containing 214 amino acid residues.

Pertuzumab (pertuzumab) is a human IgG genetically substituted with the amino acid sequence of the antigen-binding site of the mouse monoclonal antibody (2C 4) ₁ Is composed of the complementarity determining region of mouse anti-HER 2 monoclonal antibody and human IgG ₁ Is composed of framework regions and constant regions. Pertuzumab is produced by chinese hamster ovary cells. Pertuzumab is clinically used as an antitumor agent for indication of HER 2-positive breast cancer, and is available as Perjeta (registered trademark). In Perjeta (registered trademark), pertuzumab is a glycoprotein (molecular weight: about 148,000) composed of 2 molecules of an H chain (γ1 chain) containing 449 amino acid residues and 2 molecules of an L chain (κchain) containing 214 amino acid residues.

(1-3) chelating agents

In the present invention, the chelating agent is not particularly limited as long as it is a substance having a site in the structure to which a radiometal nuclides coordinates. Examples of the chelating agent include: CB-TE2A (1, 4,8, 11-tetraazabicyclo [6.6.2] hexadecane-4, 11-diacetic acid), CDTA (cyclohexane-trans-1, 2-diamine tetraacetic acid), CDTPA (4-cyano-4- [ [ (dodecylthio) thioformyl ] 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-tetra (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) tetra (methylene)) tetraphosphonic acid), DOTMP (1, 4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetra (methylenephosphonic acid)), DOTA-4AMP (1, 4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetra (acetamidomenephosphonic acid)), D02P (tetraazacyclododecane-dimethanephosphonic acid), deferoxamine (DFO), dto (N, N-bis- [2, N-carboxyethyl ] glycine ] - [ 2] pa, CHX-a "-DTPA (2, 2' - ((2- (((1 s,2 r) -2- (bis (carboxymethyl) amino) cyclohexyl) (carboxymethyl) amino) ethyl) azetidinodiyl) diacetic acid), DTPA-BMA (5, 8-bis (carboxymethyl) -11- [2- (methylamino) -2-oxoethyl ] -3-oxo-2, 5,8, 11-tetraazatridecane-13-oic acid), EDTA (2, 2',2",2' "- (ethane-1, 2-diylbis (azetidinyl)) tetraacetic acid), NOTA (1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid), NOTP (1, 4, 7-triazacyclononane-1, 4, 7-tristris (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-tetra (carboxymethyl) -3,6,9, 12-tetraazatetradecanedioic acid), HEHA (1,2,7,10,13-hexaazaoctadecane-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 "" -pentaacetic acid), H4octapa (N, N ' -bis (6-carboxy-2-pyridylmethyl) -ethylenediamine-N, N ' -diacetic acid), H2 bippa 2 (6, 6' - ({ 9-hydroxy-1, 5-bis (methoxycarbonyl) -2, 4-bis (pyridin-2-yl) -3, 7-diazabicyclo [3.3.1] nonan-3, 7-diyl } bis (-methylene)) dipicolinic acid), H2 ddpa (1, 2- {6- (carboxy) -pyridin-2-yl } -methylamino ] ethane), H2 macrppa (6- (1,4,10,13-tetraoxa-7, 16-diazacyclooctadecane-N, N ' -methyl) picolinic acid), H5decapa (N, N "-bis (6-carboxy-2-pyridylmethyl) -diethylenetriamine-N ', N, 7-diazabicyclo [3.3.1] nonan-3, 7-diyl } bis (-methylene)) dipicolinic acid), H2 decpa (1, 2- [ {6- (carboxy) -pyridin-2-yl } -methylamino ] ethane), H2 macipa (6- (1,4,10,13-tetraoxa-N, 16-diaza-N, N ' -methyl) picolinic acid), tris (N, N ' -methyl) and the like.

[ chemical formula 3]

(in the formula (A), R ₁₁ 、R ₁₂ 、R ₁₃ And R is ₁₄ Respectively and independently represent by- (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 A group consisting of COOH, R ₁₅ P is an integer of 0 to 3 inclusive and is a hydrogen atom. )

The compound represented by the formula (A) is preferably a compound containing a structure derived from 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA) or a derivative thereof, specifically, a compound containing a structure derived from a compound selected from DOTA (1, 4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid), DOTMA ((1R, 4R,7R, 10R) - α, α ', a more preferable chelating agent structure of 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 (acetamidomethylene phosphonic acid)), D02P (tetraazacyclododecane-1, 4,7, 10-tetrakis (tetramethylenephosphonic acid)), can be cited: compounds represented by the following formulas (A-1) to (A-6). The chelating agent used in the radioactive complex of the present invention is more preferably DOTA-GA (a compound represented by the formula (A-6)).

[ chemical formula 4]

The chelating agent used in the present invention is linked to the peptide via a linker (L). In the radioactive complex of the present invention, the chelating agent and the linker (L) are preferably linked by a covalent bond. Thus, in the radioactive complex of the present invention, a part of groups in the above chelating agent compound may be substituted with groups forming covalent bonds with the linker (L). For example, in the case where the chelating agent used in the present invention is a compound represented by the formula (A), R ₁₂ Or R is ₁₅ May be substituted with a group that forms a covalent bond with the linker (L). Preferably, at R ₁₂ R when substituted with a group forming a covalent bond with the linker (L) ₁₅ Is a hydrogen atom, at R ₁₂ Is formed by- (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 In the case of groups consisting of COOH, R ₁₅ A group substituted to form a covalent bond with the linker (L).

The covalent bond between the chelating agent and the linker (L) may be one that does not contain thiourea bond, and examples thereof include: carbon-carbon bonds, amide bonds, ether bonds, ester bonds, and the like.

The linkage of the chelating agent to the linker (L) is formed, for example, by reaction of the N-hydroxysuccinimide ester (NHS) group of the following formula (A-7) or (A-8) or the 2, 6-dioxotetrahydro-2H-pyranyl group of the following formula (A-9) with a primary amine of the linker (L).

[ chemical formula 5]

(1-4) antibody-modified peptides

In the present invention, the peptide is not particularly limited as long as it is a peptide that site-specifically modifies an antibody, preferably site-specifically modifies an Fc region, more preferably site-specifically modifies a lysine residue in the Fc region of an antibody. Thus, the activity of the antibody itself (antigen recognition, neutralization, complement activation and/or opsonin) can be maintained.

The peptide used in the present invention may be a chain peptide or a cyclic peptide, and is preferably a cyclic peptide. More preferably, an amino acid sequence (hereinafter, also referred to as "antibody-modified peptide") represented by the following formula (i) and composed of 13 or more and 17 or less amino acid residues is included and modified with a crosslinking agent. The peptide of formula (i) is described with the left side of the amino acid sequence shown on the paper surface showing the N-terminal side and the right side of the amino acid sequence shown on the paper surface showing the C-terminal side.

(Xa)-Xaa1-(Xb)-Xaa2-(Xc)-Xaa3-(Xd)···(i)

In the formula (i), xa, xb, xc and Xd represent a consecutive a X, a consecutive b X, a consecutive c X and a consecutive d X, respectively,

x is an amino acid residue having no one of a thiol group and a haloacetyl group in a side chain, a, b, c and d are each independently an integer of 1 to 5, and a+b+c+d.ltoreq.14, xaa1 and Xaa3 each independently represent:

amino acid residues from amino acids having a thiol group in the side chain, or amino acid residues from amino acids having a haloacetyl group in the side chain, but either Xaa1 and Xaa3 is an amino acid residue from an amino acid having a thiol group, preferably Xaa1 and Xaa3 are linked to form a ring structure,

xaa2 is a lysine residue, an arginine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, 2-amino suberic acid or diaminopropionic acid, preferably a lysine residue, and Xaa2 is modified with a crosslinking agent.

Examples of the amino acid residue that can be contained in X in the above formula (i) include: amino acid residues derived from amino acids such as glycine, alanine, phenylalanine, proline, asparagine, aspartic acid, glutamic acid, arginine, histidine, serine, threonine, tyrosine, methionine, etc., and X may be amino acid residues composed of the same kind of amino acid or amino acid residues composed of different kinds of amino acid.

The numbers of a, b, c and d in the formula (i) are not particularly limited as long as they are in the above-mentioned range, and from the viewpoint of the binding stability of the peptide to the anti-HER 2 antibody, a is preferably an integer of 1 to 3, b is preferably an integer of 1 to 3, c is preferably an integer of 3 to 5, and d is preferably an integer of 1 to 3, provided that a+b+c+d is not more than 14.

At least one of Xaa1 and Xaa3 is an amino acid residue from an amino acid having a thiol group in a side chain, which may be the same or different each. Examples of the amino acid having a thiol group in a side chain include: cysteine, homocysteine. Such an amino acid residue is preferably bonded to a thioether group via disulfide bond or via a structure represented by the following formula (4). In formula (4), the dotted line represents the binding moiety to the thioether group.

[ chemical formula 6]

Xaa1 and Xaa3 can be substituted for the combination described above, one of Xaa1 and Xaa3 being an amino acid residue from an amino acid having a thiol group on a side chain, and the other can be an amino acid residue from an amino acid having a haloacetyl group on a side chain. They may be bound via a thioether bond. The haloacetyl group is substituted at the terminal with halogen such as iodine, and the halogen is released by reaction with a thiol group in the other side chain to form a thioether bond.

Specific amino acid sequences of the antibody-modified peptide represented by the formula (i) include, for example: peptides described in WO2016/186206, WO2017/217347 and WO2018/230257 may also be used.

Preferably, the antibody-modified peptide used in the present invention is an amino acid sequence consisting of 13 to 17 amino acid residues represented by the following formula.

(X _1-3 )-C-(Xaa3’)-(Xaa4’)-H-(Xaa1’)-G-(Xaa2’)-L-V-W-C-(X _1-3 )

Wherein each X independently represents an arbitrary amino acid residue other than cysteine,

c is a cysteine residue which is a cysteine residue,

h is a histidine residue which is a group consisting of amino acids,

xaa1' is a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, 2-amino suberic acid or diaminopropionic acid,

g is a glycine residue, and the amino acid is a glycine residue,

xaa2' is a glutamic acid residue or an asparagine residue,

L is a leucine residue, and the amino acid residue,

v is a residue of valine which is the residue of formula (I),

w is a tryptophan residue which is a tryptophan residue,

xaa3' is an alanine residue, a serine residue or a threonine residue, and

xaa4' is a tyrosine residue or a tryptophan residue.

In the above formula, X at N-terminal or C-terminal _1-3 The expression (C) means that 1 to 3 amino acid residues X, which are independently arbitrary, are consecutive except for cysteine (C or Cys), and the amino acid residues constituting the amino acid residues are identical or different, preferably, are composed of sequences in which all 3 residues are different.

Among these, the amino acid sequence of the antibody-modified peptide preferably has any one of the following sequences (1) to (14), and more preferably has the following sequence (1), (2), (13) or (14). In the following amino acid sequences (1) to (14), (Xaa 2) represents a lysine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, 2-amino suberic acid or diaminopropionic acid, preferably a lysine residue, preferably (Xaa 2) is modified with a crosslinking agent, and (Xaa 1) and (Xaa 3) each represent a homocysteine residue. In the following amino acid sequences (1) to (14), amino acids other than (Xaa 1), (Xaa 2) and (Xaa 3) are denoted by single letter abbreviations.

(1)DCAYH(Xaa2)GELVWCT(SEQ ID NO:3)；

(2)GPDCAYH(Xaa2)GELVWCTFH(SEQ ID NO:4)；

(3)RCAYH(Xaa2)GELVWCS(SEQ ID NO:5)；

(4)GPRCAYH(Xaa2)GELVWCSFH(SEQ ID NO:6)；

(5)SPDCAYH(Xaa2)GELVWCTFH(SEQ ID NO:7)；

(6)GDDCAYH(Xaa2)GELVWCTFH(SEQ ID NO:8)；

(7)GPSCAYH(Xaa2)GELVWCTFH(SEQ ID NO:9)；

(8)GPDCAYH(Xaa2)GELVWCSFH(SEQ ID NO:10)；

(9)GPDCAYH(Xaa2)GELVWCTHH(SEQ ID NO:11)；

(10)GPDCAYH(Xaa2)GELVWCTFY(SEQ ID NO:12)；

(11)SPDCAYH(Xaa2)GELVWCTFY(SEQ ID NO:13)；

(12)SDDCAYH(Xaa2)GELVWCTFY(SEQ ID NO:14)；

(13)RGNCAYH(Xaa2)GQLVWCTYH(SEQ ID NO:15)；

(14)G(Xaa1)DCAYH(Xaa2)GELVWCT(Xaa3)H(SEQ ID NO:16)。

The peptide represented by the above formula (i) or the peptide having the sequences (1) to (14) is preferably such that the linker (L) is introduced at the N-terminus and the C-terminus is amidated. In addition, xaa2 of these peptides is modified with a cross-linking agent, whereby the peptides can be covalently bound to the Fc region of human IgG or rabbit IgG via the cross-linking agent. In the present specification, when referred to as "human anti-HER antibody", it means an anti-HER antibody in which a region capable of binding to an antibody-modified peptide is stored in human IgG, and preferably means an anti-HER 2 antibody in which an Fc region of human IgG is stored.

The crosslinking agent may be appropriately selected by those skilled in the art, and may be a compound having at least 2 sites capable of binding to a desired amino acid (for example, lysine, cysteine, aspartic acid, glutamic acid, 2-aminosuberic acid or diaminopropionic acid, arginine, etc.). Examples thereof are not limited, and may be mentioned: a crosslinking agent containing preferably 2 or more succinimide groups such as DSG (disuccinimidyl glutarate, bis-succinimidyl glutarate), DSS (disuccinimidyl suberate, bis-succinimidyl suberate), a crosslinking agent containing preferably 2 or more imide groups such as DMA (dimethyl adipimidate.2 HCl, dimethyl adipimidate dihydrochloride), DMP (dimethyl pimelimidate.2 HCl, dimethyl pimidate dihydrochloride) and DMS (dimethyl suberimidate.2 HCl, dimethyl suberimidate dihydrochloride), and a crosslinking agent having preferably 2 or more imide groups such as DTBP (dimethyl 3,3'-dithio bispropionimidate.2 HCl, 3' -dithiodipropyl dimethyl adipimidate dihydrochloride) and DSP (dithiobis (succinimidyl propionate), dithiobis (succinimidyl propionate)), and a crosslinking agent having SS bonds such as SBAP (3- (bromoacetamido) succinic acid succinimide ester). Crosslinking agents such as DSS or DSG containing succinimide groups react with primary amines present at the N-terminus, and thus react with DSS or DSG after blocking the N-terminus, so that only the amino group of Xaa2 can be specifically modified with DSS or DSG. For example, the peptide may be reacted with DSS or DSG after a linker (L) is introduced into the N-terminus of the antibody-modified peptide. The succinimidyl group of DSS or DSG site-specifically modifies a human anti-HER 2 antibody with a peptide by reacting with Lys248 residue or Lys246 residue, preferably Lys248 residue, according to Eu numbering in a human anti-HER 2 antibody (e.g., trastuzumab). These Lys residues are present in the Fc region of human IgG, and even for anti-HER 2 antibodies other than trastuzumab, one skilled in the art can determine equivalent Lys residues relative to the amino acid sequence of the antibody.

(1-5) Joint (L)

The linker (L) is not particularly limited as long as it can link the chelating agent and the peptide in the radioactive complex of the present invention. The linker (L) used in the present invention is not particularly limited as long as it contains a thiourea bond, and examples thereof include: substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, polyethylene glycol (PEG) based, peptides, sugar chains, disulfide groups, amide groups, combinations thereof, and the like.

In the present specification, the linker (L) is a generic term for a linker for linking the peptide-modified anti-HER 2 antibody and the chelating agent, and includes an antibody-modified linker (L ₁ ) And chelate linker (L) ₂ ) Is a term for (a). Antibody modified linker (L) ₁ ) The details of (2) are described later, which are the linker introduced to the N-terminal side of the peptide described in (1-4), and the chelate linker (L ₂ ) The details of (2) are also described below, and this is a linker introduced into the functional group of the chelating agent described in (1-3).

The linker (L) used in the present invention may comprise a binding site formed by a click reaction, preferably an antibody modified linker (L ₁ ) With chelate joints (L) ₂ ) Bonding is performed by a click reaction. In the present invention, it is preferable that the binding site formed by the click reaction does not contain thiourea bond with the chelating agent. In other words, a chelate linker (L ₂ ) Does not contain thiourea bonds. Here, the bonding site formed by the click reaction is preferably a structure containing a triazole skeleton represented by the following formula (10 a) or (10 b) or a structure containing a pyridazine skeleton represented by the following formula (10 c). Since the formula (10 a) and the formula (10 b) are in an isomeric relationship, they may be contained in any ratio.

[ chemical formula 7]

In the formula (10 a) and the formula (10 b), R _1A Represents the site of attachment to a chelating agent, R _2A Represents the site of attachment to the antibody-modified peptide. In the formula (10 c), R _3A And R is _4A One of them represents a hydrogen atom, a methyl group, a phenyl group or a pyridyl group, the other represents a linking site with a chelating agent, R _5A Represents the site of attachment to the antibody-modified peptide. In the formulae (10 a), (10 b) and (10 c), the linker to the antibody-modified peptide is via an antibody-modified linker (L ₁ ) Is linked to a peptide, and the linking site with the chelating agent is via a chelating linker (L ₂ ) A chelating agent is attached.

The radioactive complex of the present invention is obtained by complexing 1 molecule or 2 molecules of a chelator with 1 molecule of an anti-HER 2 antibody because the antibody is modified with peptide site specificity and the peptide and the chelator are linked via a linker (L).

Process for producing composite (1-6)

The method for producing a radioactive complex of the present invention can be produced by the following 2 steps: a coupling step of coupling the chelating agent with an anti-HER 2 antibody; and a complex formation step of forming a complex of the radionuclide and the chelating agent. The coupling step may be performed before or after the complex formation step.

In the coupling step, the Fc region of the antibody is site-specifically modified with a chelating agent having an antibody-modifying peptide or a linker (L) represented by the above formula (i).

In the complex formation step, the radionuclide is chelated (a complex is formed) with the chelating agent. The radiometal nuclides used herein are preferably used in an ionizable form, and more preferably in an ionic form, from the viewpoint of improving the efficiency of complex formation. The complex formation step is not limited as long as a complex with the radionuclide can be formed, and the order of adding the radionuclide to the chelating agent is not limited. 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 the formation of the complex, the resulting complex may be purified using a filter, a membrane filter, a column packed with various fillers, chromatography, or the like.

The method for producing a radioactive complex of the present invention preferably includes a coupling step after the complex formation step.

In a more preferred embodiment, in the complex formation step (a), a complex is formed between the radiometal nuclides and a chelating agent having the 1 st atomic group capable of undergoing a click reaction as a substituent for complexing with an antibody. Then, in the coupling step (B), an antibody-modified peptide represented by the above formula (i) and a workable point are used Antibody modified linker (L) to the reacted 2 nd radical ₁ ) A click reaction is performed between the peptide-modified antibody whose Fc region is site-specifically modified and the chelating agent forming a complex obtained in the step (a), to obtain the radioactive complex of the present invention.

The steps (a) and (B) are described in detail below.

As the combination of the 1 st atomic group and the 2 nd atomic group which can perform the click reaction, an appropriate combination is selected according to the type of the click reaction, and examples thereof include: combinations of alkynes with azides, combinations of 1,2,4, 5-tetrazines with alkenes, and the like. These atomic groups may be any atomic group in which the 1 st atomic group has one of the combinations of the above atomic groups and the 2 nd atomic group has one atomic group different from the 1 st atomic group of the combinations of the above atomic groups. From the viewpoint of both stability of the chelating agent and the antibody and improvement of binding efficiency thereof, the chelating linker (L ₂ ) Is alkyne and antibody modified linker (L ₁ ) Is azide, or chelate linker (L ₂ ) Is 1,2,4, 5-tetrazine and antibody modified linker (L ₁ ) Is an olefin. Specific examples of click reactions based on combinations of such atomic groups include: huisgen cycloaddition reaction or electron-withdrawing demand Diels-Alder reaction, etc.

Specific examples of combinations of atomic groups that can be subjected to the click reaction include: as shown in the following formula, the combination of an atomic group (formula (1 a)) containing Dibenzylcyclooctyne (DBCO) as an alkyne of the 1 st atomic group with an atomic group (formula (2 a)) containing azide of the azido group as the 2 nd atomic group; alternatively, the 1 st radical comprises a combination of a radical of 1,2,4, 5-tetrazine (formula (1 b)) and a radical of an alkene comprising trans-cyclooctene (TCO) as the 2 nd radical (formula (2 b)). Preferably, the combination of formula (1 a) and formula (2 a) is used.

[ chemical formula 8]

In the formula (1 a), R ₁ Represents the site of attachment to the chelating agent, R in formula (2 a) ₂ Representation and resistanceThe attachment site of the peptide is modified by the body.

[ chemical formula 9]

In the formula (1 b), R ₃ And R is ₄ One of them represents a linking site with a chelating agent or a linking site with an antibody-modified peptide, and the other represents a hydrogen atom, methyl group, phenyl group or pyridyl group. When the radical of formula (1 b) is linked to a chelating agent, R ₅ Is a linking site with an antibody-modified peptide, R is a moiety which, when the atomic group of formula (1 b) is linked to the antibody-modified peptide ₅ Represents the site of attachment to the chelating agent.

When an atomic group including Dibenzylcyclooctyne (DBCO) represented by the above formula (1 a) as an alkyne of the 1 st atomic group is used, there may be mentioned: various DBCO reagents are commercially available. Specifically, for example, DBCO-C6-acid, dibenzylcyclooctyne-amine, dibenzylcyclooctyne maleimide, DBCO-PEG acid, 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., but dibenzylcyclooctyne maleimide is preferably used.

In the complex forming step (a), more preferably, a compound having a structure represented by the following formula (ii) is used.

A-B-C···(ii)

In the formula (ii), A is the chelating agent, and B and C are collectively referred to as a chelating linker (L) ₂ )。

In the formula (ii), B is represented by the following formula (iib).

[ chemical formula 10]

In the formula (iib), la and Lb independently represent a binding linker having at least an amide bond and having a carbon number of 1 to 50, t is an integer of 0 to 30, s is 0 or 1, a binding site to a, and a binding site to C.

In the formula (ii), C is any one of an alkyne derivative represented by the following formula (iic) or a tetrazine derivative represented by the formula (iid).

[ chemical formula 11]

In the formula (iic), X is CHRk-, or N-, Y is CHRk or c=o, rk independently represents a hydrogen atom or an alkyl group having 1 or more and 5 or less carbon atoms, when X is CHRk-, Y is CHRk, the Rk moieties may together form a cycloalkyl group, rf, rg, rh, and Ri independently represent a hydrogen atom, a halogen atom, an alkyl group having 1 or more and 5 or less carbon atoms, rf and Rg together or Rh and Ri together may form a hydrocarbon ring, X represents a binding site with B, and in the formula (iid), rj represents a binding site with B, and Rj represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group).

As the chelating agent used in the step (a), more preferable is: in the above formula (A), R ₁₁ ～R ₁₄ Is- (CH) ₂ ) _p COOH, p is 1, R ₁₅ DOTA derivatives which are binding sites to B; or R is ₁₁ ～R ₁₄ Is- (CH) ₂ ) _p COOH, p is 1, R ₁₂ Is the binding site (x) with B, R ₁₅ DO3A derivatives or DOTAGA derivatives which are hydrogen atoms.

In the formula (ii), when a is DO3A, B is preferably DO3A-peg t-DBCO, wherein La is a bond having 1 to 50 carbon atoms including an amide bond, s is 0 or 1, when s is 1, t is an integer of 0 to 30 carbon atoms including an amide bond, lb is an alkyne derivative represented by the formula (iic), wherein 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 X is a bond with B.

In the formula (ii), when a is the aforementioned dotga derivative, B is preferably a dotga-peg t-DBCO derivative in which La is a bond having 1 to 50 carbon atoms including an amide bond, s is 0 or 1, when s is 1, t is an integer of 0 to 30 carbon atoms including an amide bond, lb is an alkyne derivative represented by the formula (iic) in which X is N-X, 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 X is a bond with B. More preferred is DOTAGA-DBCO described below.

[ chemical formula 12]

The lower limit of the molar ratio of the chelating agent to the radiometal nuclides is preferably 10/1 or more, more preferably 100/1 or more, still more preferably 500/1 or more, and the upper limit is preferably 10000/1 or less, more preferably 8000/1 or less, still more preferably 7000/1 or less, for example, a range of preferably 100/1 or more and 7000/1 or less, more preferably 500/1 or more and 7000/1 or less, in terms of the chelating portion/radiometal nuclides.

The complex formation reaction is preferably carried out in a solvent. As the solvent, for example, there may be used: water, physiological saline, or sodium acetate buffer, ammonium acetate buffer, phosphate buffer physiological saline, tris buffer, 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid buffer (HEPES buffer), or a buffer such as tetramethylammonium acetate buffer.

The amount of the solvent is not particularly limited, but from the viewpoint of practical use in the production process, the lower limit is in the range of 0.01mL or more, preferably 0.1mL or more, more preferably 1.0mL or more, still more preferably 10mL or more, still more preferably 100mL or more, and the upper limit is preferably 1000mL or less, more preferably 100mL or less, still more preferably 10mL or less, still more preferably 1.0mL or less, for example, 0.01mL or more and 100mL or less, at the beginning of the process (a).

The concentration of the chelating agent in the reaction liquid of the complex formation reaction is independent, and from the viewpoint of the yield of the target chelating agent, the lower limit is preferably 0.001. Mu. Mol/L or more, more preferably 0.01. Mu. Mol/L or more, still more preferably 0.1. Mu. Mol/L or more, more preferably 1. Mu. Mol/L or more, and the upper limit is preferably 1000. Mu. Mol/L or less, more preferably 100. Mu. Mol/L or less, still more preferably 10. Mu. Mol/L or less at the beginning of the step (A), for example: 1 mu mol/L or more and 100 mu mol/L or less.

The temperature of the complex forming reaction may be, for example, room temperature (25 ℃) or a temperature under heating, and from the viewpoint of both suppressing decomposition of the chelating agent and improving the efficiency of formation of the complex, the lower limit is preferably 20℃or more, more preferably 30℃or more, more preferably 35℃or more, more preferably 37℃or more, particularly preferably 45℃or more, and the upper limit is preferably 150℃or less, more preferably 120℃or less, more preferably 100℃or less, more preferably 90℃or less, for example, a range of preferably 30℃or more and 100℃or less, more preferably 35℃or more and 90℃or less.

The antibody used in the step (B) is an antibody-modified linker (L) using an antibody-modified peptide represented by the above formula (i) and a click-reaction-capable 2 nd atomic group ₂ ) A peptide-modified antibody obtained by site-specifically modifying the Fc region (constant region) of the anti-HER 2 antibody described in detail in the above-mentioned "(1-2) antibody".

The antibody-modified peptide can be produced by using amino acids (both natural amino acids and unnatural amino acids) in combination, for example, by peptide synthesis methods such as liquid phase synthesis, solid phase synthesis, automatic peptide synthesis, gene recombination and phage display. In the synthesis of the peptide, the functional group of the amino acid to be used may be protected as required. These can be carried out, for example, according to the methods described in WO2017/217347 and WO 2018/230257.

Antibody modified linker (L) ₂ ) The linker (L) may be represented by the following formula (S1) and the antibody-modified peptide ₂ ) And (3) a joint formed by combining.

*-((L _i ) _m -Z) _k -L _ii -AG2···(S1)

(wherein, represents a binding site to the N-terminus or C-terminus of a peptide,

L _i is a polyethylene glycol (PEG) joint part,

m is an integer of 1 to 50,

z is the same as (L) _i ) _m And L _ii A 2 nd joint part to be combined,

k is 0 or 1, and the number of the groups is,

L _ii for the 2 nd PEG-based linker,

AG2 is the 2 nd radical. )

In the above formula (S1), Z has a structure of (L) _i ) _m And L is equal to _ii The linker structure to be bonded to each other is not particularly limited, and may include an amino acid sequence composed of 1 to 5 amino acid residues, for example. In this case, the amino acid sequence contained in Z preferably contains a cysteine residue, and more preferably is bound to L2 via a thioether group formed by binding a thiol group of the cysteine residue to a maleimide group.

In the present invention, L is constituted by _ii The polyethylene glycol (PEG) linker of (b) preferably has a structure represented by the following formula (P2). In the formula (P2), n is an integer, preferably 1 to 50, more preferably 1 to 20, still more preferably 2 to 10, and still more preferably 2 to 6.

[ chemical formula 13]

One end of the structure of the PEG linker may be modified by a structure derived from a commercially available pegylation reagent or a structure derived from a reagent commonly used in pegylation, and examples thereof are not particularly limited: structures derived from diglycolic acid or derivatives thereof, maleimide or derivatives thereof.

Modification of linker to antibody (L) ₂ ) The method of introducing the 2 nd radical may be as follows: after obtaining an antibody-modified peptide having a desired amino acid sequence by the above method, the peptide is dissolved in a solution containing a cosolvent, a reducing agent, and an acid as needed, and then the solution is preparedAn organic solvent solution containing an azide group or a trans-cyclooctene (TCO) as the 2 nd radical is added thereto, and stirred at room temperature to introduce the 2 nd radical.

In the case of introducing an azide group-containing radical as the 2 nd radical, the azide group is directly introduced to the N-terminal or C-terminal of the peptide according to a conventional method using a commercially available azide group-introducing reagent, or the azide group-containing radical may be introduced via the above-mentioned linker structure. Examples of the azido introduction reagent to be used include: silyl azide, phosphoric acid azide, alkyl ammonium azide, inorganic azide, sulfonyl azide, PEG azide, or the like.

In addition, in the case of introducing a TCO-containing radical as the 2 nd radical, the TCO may be directly introduced to the N-terminal or C-terminal of the peptide according to a conventional method using a commercially available click chemistry reagent containing TCO, or the TCO-containing radical may be introduced via the above-described linker structure.

The method of binding the antibody-modified peptide to the human anti-HER 2 antibody to obtain the peptide-modified antibody can be carried out according to the description of WO2016/186206, for example, by dispersing the above-mentioned antibody-modified peptide, the human anti-HER 2 antibody, a crosslinking agent and a catalyst as needed in an appropriate buffer. Here, the crosslinking agent may be the one described above.

In one aspect, the present disclosure relates to a method of producing a complex of an antibody modifying peptide and a human anti-HER 2 antibody, the method comprising: and mixing the antibody-modified peptide modified with the crosslinking agent with the human anti-HER 2 antibody. Through this step, a crosslinking reaction can occur between the antibody-modified peptide modified with the crosslinking agent and the human anti-HER 2 antibody. The cross-linking reaction can occur site-specifically between the amino acid residue of Xaa2 described above of the antibody-modified peptide and Lys248 or Lys246, preferably Lys248, according to Eu numbering in human IgG Fc.

The conditions of the mixing step are not particularly limited as long as they are conditions under which a crosslinking reaction occurs between the antibody-modified peptide and the human anti-HER 2 antibody. For example, the reaction may be performed by mixing the antibody-modifying peptide and the human anti-HER 2 antibody in a suitable buffer at room temperature (e.g., about 15℃to 30 ℃). The mixing step may be performed by adding a catalyst for promoting the crosslinking reaction in an appropriate amount as required.

As an example, a solvent containing at least water is added to dissolve the human anti-HER 2 antibody. In addition to water, the solvents may be exemplified by: dimethyl sulfoxide, acetonitrile, physiological saline, or a buffer such as sodium acetate buffer, ammonium acetate buffer, phosphate buffer physiological saline, tris buffer, HEPES buffer, or tetramethylammonium acetate buffer or histidine buffer. When a buffer is used, the pH at 25 ℃ is preferably 4.0 or more and 10.0 or less, more preferably 5.5 or more and 8.5 or less, from the viewpoint of the stability of the antibody. At the beginning of the crosslinking reaction, the concentration of the antibody is preferably set to 1.0. Mu. Mol/L or more at the lower limit and 1000. Mu. Mol/L or less at the upper limit, and more preferably 500. Mu. Mol/L or less at the upper limit.

Then, the antibody-modified peptide modified by the crosslinking agent and the catalyst as needed are added, and they can be dispersed at 10℃or more and 30℃or less.

The mixing ratio of the antibody-modified peptide to the human anti-HER 2 antibody in the mixing step is not particularly limited. The molar ratio of antibody modifying peptide to human anti-HER 2 antibody can be set, for example, to 1: 1-20: 1. preferably 2: 1-20: 1 or 5:1 to 10:1.

In a preferred embodiment, in the mixing step, the antibody-modified peptide (molar ratio) may be mixed with respect to the human anti-HER 2 antibody at a ratio of 0.5 to 2.2, preferably 0.8 to 1.8. In this way, an antibody (hereinafter, referred to as "monovalent antibody") in which 1 molecule of the antibody-modified peptide binds to 1 molecule of the human anti-HER 2 antibody can be efficiently obtained.

The mixing time (reaction time) in the mixing step is not limited as long as the crosslinking reaction between the antibody-modified peptide and the human anti-HER 2 antibody occurs, and may be, for example, 1 minute to 5 hours, preferably 10 minutes to 2 hours.

The peptide-modified antibody obtained in the above steps is a mixture of an antibody (i.e., monovalent antibody) obtained by binding 1 molecule of the antibody-modified peptide to 1 molecule of the human anti-HER 2 antibody and an antibody (hereinafter referred to as "divalent antibody") obtained by binding 2 molecules of the antibody-modified peptide to 1 molecule of the human anti-HER 2 antibody in an arbitrary ratio, and may be directly supplied to the subsequent step, or may be obtained by separating and purifying the unmodified antibody, the monovalent antibody and the divalent antibody by using a filter, a membrane filter, a column packed with various fillers, various chromatographs, or the like, and then supplying only any valence of the antibody to the subsequent step. As a result of purification, when an unmodified antibody and an antibody of another valence cannot be separated, the mixture containing the unmodified antibody and the antibody may be supplied to the subsequent step.

In the case of separating and purifying an unmodified antibody, a monovalent antibody and a divalent antibody, the separation and purification can be performed by any of the above purification methods, and a column packed with various fillers can be used, for example, a column packed with a filler in which a protein such as protein a, protein G or the above antibody-modified peptide is bound to a carrier can be used. The shape of the carrier of the filler packed in such a column may be: examples of the material of the carrier include a gel (for example, a column gel), a particle, a bead, a nanoparticle, a microparticle, and a large bead: magnetic substances, latex, agarose, glass, cellulose, sepharose, nitrocellulose, polystyrene, other polymeric materials. Specifically, examples may be: an IgG-BP column obtained by binding the above antibody-modified peptide to a column gel (see WO 2021/080008).

The IgG-BP column is a column obtained by immobilizing an IgG-binding peptide. Since the binding site of the bivalent antibody is already occupied by the IgG-binding peptide, it cannot bind to the column, and only the monovalent antibody exhibits affinity to the column. Using the IgG-BP column, the unmodified antibody and the first antibody composition containing relatively more monovalent antibodies and the second antibody composition containing relatively more bivalent antibodies can be separated and purified, respectively, by utilizing the difference in interaction between the respective antibody-modified peptides. In a preferred embodiment, the molar ratio of unmodified antibody to monovalent antibody in the first antibody composition is from 4 to 47:53 to 96, preferably 4 to 30:70 to 96, more preferably 4 to 20:80 to 96, more preferably 4 to 10: 90-96 percent.

In this way, the isolated and purified first antibody composition or second antibody composition can be used as it is for the click reaction in the subsequent step (B), or can be used for the click reaction in the step (B) after the protein concentration of the peptide-modified antibody contained therein is adjusted.

The click reaction in the step (B) is performed between a first group capable of performing a click reaction, which is included in the chelating agent, and a second group capable of performing a click reaction, which is included in the peptide-modified antibody. By such a click reaction, a binding group (a substituent capable of complexing with an antibody) linking the chelating agent and the antibody is formed.

The order of addition of the peptide-modified antibody and the complex obtained in step (a) is not limited as long as the peptide-modified antibody and the complex can be subjected to click reaction, and for example, one of the complex and the peptide-modified antibody may be added to a reaction vessel containing a solvent and then the other may be added to the reaction vessel to react, or the other may be added to a dispersion obtained by dispersing one of the chelating agent and the antibody in a solvent to react. Alternatively, these may be added simultaneously to a reaction vessel containing a solvent to perform the reaction.

As the solvent used for the click reaction in the step (B), a solvent including water may be used, and for example, may be used: water, physiological saline, or a buffer such as sodium acetate buffer, ammonium acetate buffer, phosphate buffer physiological saline, tris buffer, HEPES buffer, or tetramethylammonium acetate buffer. When a buffer is used, the pH at 25 ℃ is preferably 4.0 or more and 10.0 or less, more preferably 5.5 or more and 8.5 or less, from the viewpoint of both stability of the complex and the antibody and their binding efficiency.

The amount of the reaction liquid is not particularly limited, but from the viewpoint of practical use in the production process, the lower limit is preferably 0.001mL or more, more preferably 0.01mL or more, still more preferably 0.1mL or more, still more preferably 1mL or more, and the upper limit is preferably 1000mL or less, more preferably 100mL or less, still more preferably 10mL or less, still more preferably 1mL or less, for example, the range of preferably 0.001mL or more and 1000mL or less, and the range of preferably 0.1mL or more and 10mL or less at the beginning of the process (B).

The concentrations of the chelating agent and the antibody in the reaction solution are independent from each other, and at the beginning of the step (B), the lower limit is preferably 0.001. Mu. Mol/L or more, more preferably 0.01. Mu. Mol/L or more, still more preferably 0.1. Mu. Mol/L or more, still more preferably 1.0. Mu. Mol/L or more, and the upper limit is preferably 1000. Mu. Mol/L or less, more preferably 100. Mu. Mol/L or less, for example, the range of 0.1. Mu. Mol/L or more and 1000. Mu. Mol/L or less, and from the viewpoint of the yield of the target complex, the range of 1. Mu. Mol/L or more and 100. Mu. Mol/L or less is more preferable.

In view of preventing undesired denaturation of the antibody and improving the reaction efficiency, the upper limit of the reaction temperature is preferably 50℃or less, more preferably 40℃or less, in the click reaction in the step (B). The lower limit of the reaction temperature is not particularly limited as long as the reaction proceeds, and is preferably 15℃or higher. The reaction time of the click reaction is preferably 5 minutes or more, more preferably 10 minutes or more, still more preferably 24 hours or less, still more preferably 20 hours or less, for example, preferably 5 minutes or more and 24 hours or less, still more preferably 10 minutes or more and 20 hours or less, based on the reaction temperature described above.

The resulting complex may be used as it is, or may be purified by using a filter, a membrane filter, a column packed with various fillers, chromatography, or the like.

The complex produced in steps (a) and (B) is a complex in which a lysine residue in the Fc region of an anti-HER 2 antibody is specifically modified with a chelating agent. The complex has 1 molecule or 2 molecules of the chelating agent described above relative to 1 molecule of the antibody. The chelator site-specifically modifies the Fc region of the antibodies of the invention via the linker (L). The linker (L) is composed of a chelating linker (L) ₂ ) And the joint (L) ₂ ) The 1 st atomic group connected, the 2 nd atomic group capable of click reaction with the 1 st atomic group, and the antibody-modified linker (L) connected with the 2 nd atomic group ₁ ) (comprising the antibody-modified peptide represented by the above formula (i)). Thus, the linker (L) has a chemical structure from the 1 st and 2 nd radicals. As suchThe chemical structure is considered to be a structure containing a triazole skeleton represented by the above formula (10 a) or (10 b) or a structure containing a pyridazine skeleton represented by the following formula (10 c). Since the formula (10 a) and the formula (10 b) are in an isomeric relationship, they may be contained in any ratio.

(1-7) radiopharmaceuticals (radiopharmaceutical (1))

A radiopharmaceutical refers to a composition comprising the radioactive complex of the present invention and formulated into a form suitable for administration into a subject's living body. The radiopharmaceutical can be produced, for example, by dissolving the radioactive complex produced by the method described in (1-6) above directly or after purifying it in a solvent mainly composed of water and substantially isotonic with a living body. In this case, the radiopharmaceutical is preferably in the form of an aqueous solution, and may contain other pharmaceutically acceptable ingredients, as required. Radiopharmaceuticals are administered orally or parenterally, e.g., intravenously, subcutaneously, intraperitoneally, intramuscularly, etc., in effective amounts to organisms for the treatment of cancer, diagnosis of cancer, or detection of lesions, etc.

Here, examples of the administration target include animals such as humans, mice, rats, monkeys, guinea pigs, chimpanzees, sheep, goats, dogs, cats, pigs, cows, and horses, and the like, and are not particularly limited. Preferably a human.

Preferable diseases to be treated include: HER2 overexpressing cancers. The type of cancer to be treated, diagnosed or detected in the present invention, in which HER2 is overexpressed, is not particularly limited as long as HER2 is overexpressed, and examples thereof include: salivary gland cancer, ovarian cancer, bladder cancer, cholangiocarcinoma, gastric cancer or breast cancer. In addition, HER2 overexpressing cancers may be any stage of disease, either localized or metastatic, or primary or recurrent. Here, "overexpression" means a state in which significant amplification of HER2 gene or significant increase in expression of HER2 protein in tumor tissue compared to non-tumor tissue is observed when measured by a known examination method.

An "effective amount" as used herein refers to an amount that achieves a diagnostic or therapeutic effect in a subject. The effective amount to be administered to a subject varies depending on the kind of the subject, the body weight of the subject, the dosage form (tablet, injection, etc.) and the route (oral administration, parenteral administration, etc.) of administration, and the severity of the disease (e.g., cancer), etc. The physician or veterinarian can consider these factors in determining the appropriate effective amount.

The radiopharmaceutical of the present invention has a radiochemical purity of a certain proportion or more at a time point during which a multiple of 1 to 5 times or less of the half-life of the radionuclide constituting the radiopharmaceutical passes, based on the half-life of the radionuclide constituting the radiopharmaceutical when stored at room temperature. When the radionuclide is a beta-ray nuclide (for example, lu-177 or Y-90), the radiochemical purity of the complex is preferably 90% or more, more preferably 95% or more, when stored at room temperature for 7 days from the end of production. In the case where the radionuclide is an α -ray nuclide (for example, ac-225), the radiochemical purity of the complex is preferably 90% or more, more preferably 95% or more, when stored at room temperature for 14 days from the end of production. Here, the "room temperature" in the present specification preferably means "normal temperature" defined in japanese pharmacopoeia, specifically, means 15 to 25 ℃.

Here, the radiochemical purity refers to a percentage of radioactivity (count) corresponding to a peak of a complex to total radioactivity (count) detected in the case of analyzing a sample using a commercially available radiation detector. High performance liquid chromatography or thin layer chromatography may be employed in the radiochemical purity analysis, but thin layer chromatography is preferably employed. More preferably, thin layer chromatography using the conditions described in examples described below is used.

As described above, the radiopharmaceutical of the present invention is preferably in the form of an aqueous solution, but from the viewpoint of maintaining radiochemical purity, the form of a buffer is more preferable. The buffer may be any buffer used in an antibody drug containing an anti-HER 2 antibody or an ADC of an anti-HER 2 antibody as an active ingredient, and is not particularly limited, and histidine buffer or succinic buffer may be used as an example. The histidine buffer is composed of histidine and a salt thereof, and for example, may be composed of histidine and a hydrochloride thereof, or histidine and an acetate thereof. The succinic acid buffer is composed of succinic acid and salts thereof, for example, succinic acid and sodium salts thereof. The radiopharmaceuticals of the present invention may contain any saccharide such as sucrose or trehalose, and may also contain a solubilizing agent such as polysorbate 20 or polysorbate 80.

As the radionuclide, the radiopharmaceutical of the present invention can be used for the radiation therapy of cancer by selecting a radionuclide having a therapeutic effect, specifically, selecting an alpha-emitting radionuclide or a beta-emitting radionuclide (preferably Ac-225, Y-90, lu-177, more preferably Ac-225). In this radiotherapy for internal radiation, the radiopharmaceutical of the present invention may be administered by intravenous injection or oral administration, and the radioactive complex of the present invention may be accumulated at a focal site such as a primary cancer site or a metastatic site, and cancer cells at the focal site may be destroyed by radiation emitted from a radionuclide. The amount and dose of the radiopharmaceutical of the present invention to be administered are appropriately selected according to the availability of the active ingredient, the mode and route of administration, the stage of progression of cancer, the size, weight and age of the patient, and the type or amount of the therapeutic agent for other diseases to be used in combination.

In addition, by selecting a positron-emitting radionuclide or a gamma-emitting radionuclide (preferably Zr-89) as the radionuclide, it is possible to use for diagnosis of cancer or detection of lesions. The use of a radiopharmaceutical with a positron-emitting radionuclide is suitable for PET (Positron Emission Tomography: positron emission tomography) examination, and the use of a radiopharmaceutical with a gamma-emitting radionuclide is suitable for SPECT (Single Photon Emission Computed Tomography: single photon emission computed tomography) examination. It can be used in combination with the diagnosis of cancer or detection of lesions in the above-mentioned radiotherapy for cancer. The radiopharmaceutical for cancer diagnosis of the present invention can be used for the diagnosis of cancer before and after the administration of an internal radiotherapy. The present invention can be used for diagnosis of cancer before the administration of the radiotherapy, and for determining whether or not to perform a therapeutic selection of the radiotherapy for cancer using the radiopharmaceutical of the present invention including the metal nuclide emitting the α -ray. Further, the present invention can be used for diagnosis of cancer after the administration of the radiotherapy, and for determining whether or not the treatment plan such as the effect of the radiotherapy of cancer using the radiopharmaceutical of the present invention and the increase or decrease in the dose is appropriate.

(2) Radiopharmaceutical (2)

Another scheme of the invention is as follows: the complex containing a chelator chelated with a radionuclide and an anti-HER 2 antibody as an active ingredient, wherein the anti-HER 2 antibody and the chelator are linked without thiourea bonds, and the complex has a radiochemical purity of a certain proportion or more at a time point during a multiple of 1 to 5 times the half-life of the radionuclide constituting the radiopharmaceutical when stored at room temperature. When the radionuclide is a beta-ray nuclide (for example, lu-177 or Y-90), the radiochemical purity of the complex is preferably 90% or more, more preferably 95% or more, when stored at room temperature for 7 days from the end of production. When the radionuclide is an α -ray nuclide (for example, ac-225), the radiochemical purity of the complex is preferably 90% or more, more preferably 95% or more, when stored at room temperature for 14 days from the end of production. The definition of room temperature is the same as for radiopharmaceutical (1) described above.

In the radiopharmaceutical (2), the method (a) to (d) below may be adopted in addition to the method using site-specific modification of the peptide in complexing the chelator with the anti-HER 2 antibody, and the same as in the radiopharmaceutical (1) except for the following method, and therefore, the description thereof will be omitted.

Method (a): a thiol (SH) group produced by partially reducing disulfide bonds (SS bonds) between polypeptide chains located at the hinge portion of an antibody is modified with a chelating agent or linker (L) having a maleimide group reactive with SH groups;

method (b): using a chelator or linker (L) having a maleimide group, a cysteine newly introduced into an antibody by a genetic engineering-based amino acid mutation is modified;

method (c): modification of the azido group of lysine azide newly introduced into an antibody by genetic engineering-based amino acid mutation using a chelator or linker (L) having alkyne (e.g., dibenzocyclooctyne: DBCO) with click reaction;

method (d): glutamine introduced into a specific position of an antibody by transglutaminase is modified with a chelating agent having a side chain of lysine or a linker (L).

In the present invention, since the peptide and the chelator of the anti-HER 2 antibody are site-specifically modified by ligation without using thiourea bond, a radioactive complex and a radiopharmaceutical which are stable even at room temperature can be prepared. The site-specific modification of antibodies, unlike random modification, can comprise monovalent antibodies or divalent antibodies or both in any proportion, thus becoming a stable quality radioactive complex and radiopharmaceutical. The radioactive complex of the present invention maintains the same efficacy as before. Therefore, according to the present invention, it is possible to provide a complex of an anti-HER 2 antibody and a radiopharmaceutical thereof, which have more excellent quality while maintaining the efficacy.

The technical idea of the invention also comprises the following schemes.

[1] A complex of an anti-HER 2 antibody site-specifically modified with a peptide and a chelating agent,

the radionuclides are sequestered in the chelating agent,

the peptide is linked to the chelating agent via a linker (L), and the linker (L) does not contain thiourea bond.

[2] The complex according to [1], wherein the chelating agent is DOTAGA (α - (2-carboxyethyl) -1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid).

[3] The complex according to [1] or [2], wherein the peptide is an amino acid sequence comprising 13 to 17 amino acid residues represented by the following formula (i):

(Xa)-Xaa1-(Xb)-Xaa2-(Xc)-Xaa3-(Xd)···(i)

x is an amino acid residue having no thiol group or haloacetyl group in the side chain,

a. b, c and d each independently represent an integer of 1 to 5 inclusive and satisfy a+b+c+d.ltoreq.14,

xaa1 and Xaa3 each independently represent:

an amino acid residue derived from an amino acid having a thiol group in a side chain, or an amino acid residue derived from an amino acid having a haloacetyl group in a side chain, wherein any of Xaa1 and Xaa3 is an amino acid residue derived from an amino acid having a thiol group,

Xaa1 and Xaa3 are joined to form a ring structure,

xaa2 is a lysine residue, an arginine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, 2-amino suberic acid, or diaminopropionic acid, and Xaa2 is modified with a crosslinking agent.

[4] The complex according to any one of [1] to [3], wherein the radionuclide is Ac-225, Y-90, lu-177 or Zr-89.

[5] The complex according to any one of [1] to [4], wherein the linker (L) comprises the formula (10 a), the formula (10 b) or the formula (10 c),

[ chemical formula 14]

In the formula (10 a) and the formula (10 b), R _1A Represents the junction with the chelating agent, R _2A Represents the junction with the peptide, R in the formula (10 c) _3A And R is _4A One of them represents a hydrogen atom, a methyl group, a phenyl group or a pyridyl group, the other represents a linking site with the chelating agent, R _5A Represents the site of attachment to the peptide.

[6] The radioactive complex of [5], wherein a polyethylene glycol group is contained between a junction with the peptide and the peptide.

[7] The radioactive complex of [1] to [6], wherein an anti-HER 2 antibody specifically modified with the peptide having an azide group introduced at the N-terminus is complexed by a click reaction with a radioactive metal complex of dotga-DBCO represented by the following formula:

[ chemical formula 15]

[8] The complex according to any one of [1] to [7], wherein the anti-HER 2 antibody is trastuzumab.

[9] The complex according to any one of [1] to [7], wherein the anti-HER 2 antibody is pertuzumab.

[10] A radiopharmaceutical comprising the complex according to any one of [1] to [9] as an active ingredient.

[11] The radiopharmaceutical of [10] which is used for the radiotherapy of cancer.

[12] The radiopharmaceutical of [10] for diagnosis of cancer.

[13] The radiopharmaceutical of [12] which is used in combination with an intra-radiotherapy for cancer using the radiopharmaceutical of [11 ].

[14] A radiopharmaceutical comprising, as an active ingredient, a complex of a chelator chelated with a radiometal nuclides and an anti-HER 2 antibody,

no thiourea bond is involved in the attachment of the anti-HER 2 antibody to the chelator,

the radiochemical purity of the complex is 90% or more when stored at room temperature for 7 days.

[15] The radiopharmaceutical of [14], wherein the complex is any one of [1] to [9 ].

[16] The radiopharmaceutical of [15] which is used for the radiotherapy of cancer.

[17] The radiopharmaceutical of [15] which is useful for diagnosis of cancer.

[18] The radiopharmaceutical of [17] which is used in combination with an intra-radiotherapy for cancer using the radiopharmaceutical of [16 ].

[19] A radiopharmaceutical comprising, as an active ingredient, a complex of a chelator chelated with a radiometal nuclides and an anti-HER 2 antibody,

and satisfies the following condition (1) or (2):

(1) The radionuclides mentioned above ¹⁷⁷ Lu or ⁹⁰ Y, the radiochemical purity of the complex is 90% or more when stored for 7 days at room temperature;

(2) The radionuclides mentioned above ²²⁵ Ac, the radiochemical purity of the complex was 90% or more when stored at room temperature for 14 days.

[20] A radiopharmaceutical comprising, as an active ingredient, a complex of a chelator chelated with a radiometal nuclides and an anti-HER 2 antibody,

the radiochemical purity of the complex is 90% or more at a time point when a multiple of 1 to 5 times or less of the half-life of the radionuclide is passed.

Examples

The present invention will be described in further detail with reference to examples. However, the scope of the present invention is not limited to these examples. In the following table, the "-" column indicates that no implementation is performed.

Example 1]Using ²²⁵ Preparation of composite of trastuzumab by Ac marked DOTAGA-DBCO

(1. Antibody modification step)

According to the method described in WO2017/217347, a peptide having 17 amino acid residues represented by the following formula (P3) was obtained. The amino acid sequence of the peptide is the same as that of the peptide shown in SEQ ID NO (2), xaa2 is lysine residue, and the amino group at the tail end of the side chain of the lysine residue is R ₁ The structural modifications shown. In addition, disulfide bonding was performed by 2 cysteine residues, and the N-terminus of the peptide was linked via a linker (L ₁ ) An azide group is bonded to the structure of the group containing an azide group as the 2 nd group.

[ chemical formula 16]

(SEQ ID NO:17)

(in the 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, phe represents phenylalanine.)

The peptide and trastuzumab (registered trademark) were mixed in 0.02mol/L sodium acetate buffer (pH 6.0) and the resulting mixture was allowed to react at room temperature for 30 minutes to obtain a solution containing the peptide-modified antibody. The peptide-modified antibody is obtained by site-specifically modifying the Fc region of the antibody with the above-described peptide.

Then, the IgG-BP column was subjected to liquid chromatography, and separated into a first antibody composition containing a relatively large amount of unlabeled antibody and monovalent antibody and a second antibody composition containing a relatively large amount of bivalent antibody.

The solution containing the peptide-modified antibody obtained in the above step was diluted with 0.02mol/L sodium acetate buffer (pH 6.0), added to the IgG-BP column, and 0.10mol/L sodium acetate buffer (pH 5.7) containing 0.15mol/L sodium chloride was introduced to collect the second antibody composition, and the concentration was adjusted so that the concentration of the divalent antibody contained in the collected fraction became 15mg/mL. Thereafter, a 0.10mol/L sodium acetate buffer (pH 3.5) containing 0.15mol/L sodium chloride was introduced into the IgG-BP column, and the first antibody composition was recovered, and the concentration of the monovalent antibody contained in the recovered fraction was adjusted to 15mg/mL. The obtained solution containing the first antibody composition is supplied to a labeling step described later.

(2. Complex formation step)

According to Bernhard et al DOTAGA-anhydride: a Valuable Building Block for the Preparation of DOTA-Like Chelating Agents the method described in chem.Eur.J.2012,18,7834-7841 produces DOTAGA-DBCO represented by the following formula. Chelating the chelateThe mixture was dispersed in 0.1mol/L sodium acetate buffer (pH 6.0) as a solvent to prepare a dispersion containing 1.7mmol/L chelating agent. Mixing 0.005mL of the dispersion, 0.075mL of 0.1mol/L sodium acetate buffer (pH 6.0), and 1.6 to 1.7MBq (calculated from the attenuation of the radioactive energy at the test day) as a radioactive metal source ²²⁵ The reaction solution obtained by reacting Ac ion solution (0.2 mol/L hydrochloric acid aqueous solution, radioactivity concentration of 320-340 MBq/mL, prepared by Oak Ridge National Laboratory, liquid amount of 0.005 mL) under heating condition, thereby obtaining ²²⁵ Ac complex solution. The molar ratio of chelating agent to radioactive metal ion is chelating agent: ²²⁵ ac ion = about 2500:1, the heating temperature of the reaction solution was 70℃and the heating time was 30 minutes.

[ chemical formula 17]

The obtained product was measured by the following method ²²⁵ Radiochemical purity (RCP) of Ac complexes. I.e. will be a part of ²²⁵ The Ac complex solution was developed by thin layer chromatography (MODEL: SGI0001, developing solvent: acetonitrile/water mixture (volume ratio: 1:1)) and then measured by using a radioactive gamma-TLC analyzer (MODEL GITA Star, manufactured by rayest). The percentage of the radioactivity (count) of the peak detected near the origin to the total radioactivity (count) detected is taken as ²²⁵ RCP (%) of Ac complex. As a result of this, the product, ²²⁵ the RCP of the Ac complex was 90%. The obtained ²²⁵ The Ac complex solution was directly used in the labeling procedure.

(3. Marking step)

Subjecting the crude product obtained in the step (2) to a purification step ²²⁵ The Ac complex solution and the peptide-modified antibody (monovalent antibody) -containing solution obtained in the step (1) were added to 0.1mol/L histidine buffer (pH 6.0) containing 0.1mol/L arginine, and a click reaction was performed at 37℃for 120 minutes, thereby obtaining ²²⁵ The Ac complex labels the antibody. ²²⁵ The amount of Ac complex and the amount of peptide modified antibody (monovalent antibody) were 85nmol, respectively, and the molar ratio of dbco groups to azide groups was about 1, respectively: 1.2. without purification ²²⁵ The reaction rate (%) of the Ac complex-labeled antibody is shown in table 1 below. Here, the reaction rate (%) refers to the labeling rate (%) in the complex formation step ²²⁵ The Ac complex labels RCP (%) of an antibody, and the labeling rate (%) refers to the amount of radioactive energy relative to the filler ²²⁵ Radioactivity (%) of Ac complex.

Reacting at 37 ℃ for 2 hours ²²⁵ The solution of the Ac complex-labeled antibody was purified using an ultrafiltration filter (manufactured by Merck Co., ltd., model: UFC 505096). Purified ²²⁵ The yields of RCP and Radiochemistry (RCY) of the Ac complex-labeled antibodies are shown in table 1 below.

²²⁵ The methods for determining RCP and RCY of the Ac complex labeled antibody are as follows. That is, thin layer chromatography (MODEL: SGI0001, a mixed solution of acetonitrile in an developing solvent of 0.1 mmol/LETTA solution (volume ratio 1:1)) was measured using a radioactive gamma-TLC analyzer (manufactured by raytest Co., ltd., MODEL GITA Star) and the percentage of the radioactivity (counted) of the peak detected near the origin to the total radioactivity (counted) detected was used as RCP (%). In addition, the radioactivity recovered after ultrafiltration purification (radioactivity calculated from the count measured by a gamma-ray spectrometer) and the total radioactivity applied at the beginning of the labeling step (from a gamma-ray spectrometer (Ge semiconductor detector: GMX10P4-70 (manufactured by ORTEC Co.), a multichannel analyzer: M7-000 (Seiko EG) &Manufactured by G company), data processing: spectrum Navigator: DS-P300 (Seiko EG)&Manufactured by G corporation) and Gamma Studio: DS-P600 (Seiko EG)&Manufactured by G corporation)) as the RCY (%).

TABLE 1

Example 2]Using ⁸⁹ Zr marked DOTAGA-DBCO for manufacturing trastuzumab compoundObject (1. Complex formation Process)

DOTAGA-DBCO was dispersed in DMSO as a solvent to prepare a dispersion containing 0.33mmol/L chelating agent. Mixing 0.030mL of the dispersion with 60MBq as a radioactive metal source ⁸⁹ A reaction solution obtained by reacting a solution of Zr ions (0.1 mol/L aqueous hydrochloric acid solution, a radioactivity concentration of 181MBq/mL, prepared by Medi-Physics Co., ltd., liquid amount of 0.33 mL) under heating to obtain ⁸⁹ Zr complex solution. The molar ratio of chelating agent to radioactive metal ion is chelating agent: ⁸⁹ zr ion = about 250:1, the heating temperature of the reaction solution was 70℃and the heating time was 60 minutes.

The obtained product was measured by the following method ⁸⁹ RCP of Zr complex. I.e. will be a part of ⁸⁹ The Zr complex solution was developed by thin layer chromatography (model: SGI0001, developing solvent: acetonitrile/water mixed solution (volume ratio: 1:1)) and then measured by using a radioactive gamma-TLC analyzer (manufactured by raytest Co., ltd., MODEL GITA Star PS). The percentage of the radioactivity (count) of the peak detected near the origin to the total radioactivity (count) detected is taken as ⁸⁹ RCP (%) of Zr complex. As a result of this, the product, ⁸⁹ the RCP of the Zr complex was 90%. The obtained ⁸⁹ The Zr complex solution was directly used in the labeling procedure.

(2. Marking step)

Subjecting the unpurified state obtained in the step (1) ⁸⁹ The Zr complex solution and the peptide-modified antibody (monovalent antibody) -containing solution obtained in the same manner as in example 1 were each added in an unpurified state to 0.1mol/L histidine buffer (pH 6.0) containing 0.1mol/L arginine, and a click reaction was carried out at 37℃for 120 minutes, thereby obtaining example 2 ⁸⁹ The Zr complex labels the antibody. ⁸⁹ The amount of Zr complex and the amount of peptide modified antibody (monovalent antibody) were 100nmol, respectively, and the molar ratio of dbco to azide was about 1, respectively: 1. example without purification ⁸⁹ The reaction rate (%) of the Zr complex labeled antibody is shown in table 2 below. Here, the reaction rate (%) is a percentage (%o) relative to the labeling rate in the complex formation step) A kind of electronic device ⁸⁹ The Zr complex labels RCP (%) of the antibody, and the labeling rate (%) refers to the amount of radioactive energy relative to the filler ⁸⁹ Radiant energy (%) of Zr complex.

The reaction was carried out at 37℃for 2 hours ⁸⁹ The solution of the Zr complex labeled antibody was purified using an ultrafiltration filter (manufactured by Merck Co., ltd., model: UFC 505096). Purified ⁸⁹ The RCP and RCY of the Zr complex labeled antibodies are shown in Table 2 below.

⁸⁹ The measurement methods of RCP and RCY of the Zr complex labeled antibody were performed in the same manner as in example 1.

TABLE 2

Comparative example 1]Using ²²⁵ Complex of trastuzumab produced by Ac-labeled DOTA-DBCO

The procedure of example 1 was followed, except that DOTAGA-DBCO was changed to DOTA-DBCO described below. The results are shown in Table 3.

[ chemical formula 18]

TABLE 3

EXAMPLE 3 formulation procedure

A portion of each of the radioactive complexes produced in accordance with the descriptions of example 1 and comparative example 1 was extracted into a 0.5mL microcentrifuge tube (manufactured by LoBind, eppendorf Co.) and diluted with a preservation buffer (19 g/L of trehalose hydrate, 0.47g/L of L-histidine hydrochloride hydrate, 0.30g/L of L-histidine and 85mg/L of polysorbate 20 mixture).

[ evaluation 1] stability evaluation

The respective radioactive complexes obtained in example 3 were stored at room temperature (24.5 to 25.5 ℃) for 2 weeks, and the RCP, the proportion of aggregates, and the antigen binding activity were evaluated at each time point (at 0 day, at 1 day, at 7 days, and/or at 14 days). In the case where the radionuclide is Ac-225, 14 days from the end of the production corresponds to about 1.5 half-lives.

[ evaluation 1-1] radiochemical purity

The RCP was analyzed by Thin Layer Chromatography (TLC). The conditions for TLC were the same as those used in the investigation of the reaction rate in example 1.

The results are shown in Table 4.

TABLE 4

When the radioactive complex without thiourea bond produced as described in example 1 was stored at room temperature for 7 days after completion of production, 99% or more in terms of RCP was maintained. When the resin composition is stored at room temperature for 14 days after completion of production, the resin composition is maintained at 99% or more in terms of RCP.

When the radioactive complex containing thiourea bond produced as described in comparative example 1 was stored at room temperature for 7 days after completion of production, 90% or more but less than 95% in terms of RCP was maintained. In the case of storage at room temperature for 14 days after completion of production, the content was less than 80% in terms of RCP.

[ evaluation 1-2] proportion of aggregates

The proportion of aggregates was confirmed by Size Exclusion Chromatography (SEC). Analysis was performed under the following conditions using a 2695 type separation module manufactured by Waters company or an e2695 type separation module as a liquid chromatography apparatus and a 2489 type UV/Vis detector manufactured by Waters company as a UV detector. The proportions of the components in the case of storage for 14 days after completion of production are shown in Table 5. When the composition was stored for 14 days after completion of the production, the proportion of the aggregates of the radioactive complex containing no thiourea bond produced as described in example 1 was reduced as compared with the proportion of the aggregates of the radioactive complex containing thiourea bond produced as described in comparative example 1.

TABLE 5

	Proportion of main peak (%)	Peak ratio of aggregate (%)
			Radioactive complexes (example 1)	94.79	1.51
Radioactive complex (comparative example 1)	93.18	3.34

[ HPLC conditions ]

Column: TOSOH TSKgel protective column SWXL (6 mm. Times.4 cm), TOSOH TSKgel G3000SWXL (5 μm, 7.8X30 cm). Times.2 (in tandem);

column temperature: a certain temperature around 25 ℃;

mobile phase: phosphate buffer (pH 6.8) containing 0.2mol/L arginine hydrochloride, 0.1 mol/L;

flow rate: 1.0 mL/min;

area measurement range: 30 minutes;

detection wavelength: 280nm.

[ evaluation of 1-3] antigen binding Activity

Antigen binding Activity in vitroAutoradiography (ARG) was confirmed (only on the day of manufacture (day 0) and the last day of storage (day 14)). SK-OV-3 cells purchased from ATCC (American Type Culture Cullection) as a HER 2-positive human ovarian cancer cell line and MDA-MB-231 cells as a HER 2-negative human breast cancer cell line were each used at 5X 10 ⁶ Individual cells and 1X 10 ⁷ Tumor-bearing mice were prepared by subcutaneously administering individual cells to the flank of female BALB/c nu/nu mice. Afterwards, SK-OV-3 tumor and MDA-MB-231 tumor were removed, and frozen sections were prepared by embedding in Tissue-Tek O.C.T.Compound (Sakura Finetek Japan). The radioactive complexes obtained in example 1 and comparative example 1 were added to PBS containing 10% bovine serum albumin to 1KBq/mL, and SK-OV-3 tumor sections and MDA-MB-231 tumor sections were immersed, respectively. After the slice is brought into contact with the imaging plate, the radiation energy combined with the slice is evaluated by reading using a scanner-type image analysis device. The results are shown in FIG. 3.

The specificity of each radioactive complex for HER2 was confirmed by evaluating the same as that of the solution obtained by adding trastuzumab to each solution.

At the final storage point (14 days), HER2 binding activity was confirmed in the radioactive complexes prepared as described in example 1 and comparative example 1. The radioactive complexes prepared as described in example 1 and comparative example 1 each bound only to SK-OV-3 tumor sections, showing HER2 selective binding. Binding to SK-OV-3 tumor sections was inhibited in solutions with trastuzumab added, confirming the specificity of bound HER 2. At the final end of storage (14 days), binding selectivity for HER2 was maintained in all samples. The radioactivity bound to the SK-OV-3 tumor slices was increased in the sample using the radioactive complex manufactured according to the description of example 1, compared to the radioactive complex manufactured according to the description of comparative example 1.

[ evaluation 2] evaluation of drug efficacy

A subcutaneous tumor-bearing model of SK-OV-3 cells was prepared using mice, and the antitumor effect of the radioactive complex prepared as described in example 1 and comparative example 1 was confirmed.

Will be purchased from ATCC as HER2 cation SK-OV-3 cells of the human ovarian carcinoma strain were suspended in McCoy's 5A medium (gibco) and subcutaneously administered 5X 10 to the flank of a 5-week-old female BALB/c nu/nu (Charles River, japan) ⁶ Tumor-bearing mice were prepared from individual cells. Tumor volume of about 100 to 300mm was confirmed 3 weeks after tumor-bearing treatment ³ The individuals fit to the shape of the tumor diameter were randomly grouped. The tumor volumes and body weights of the mice at this time are shown in Table 6. Tumor volume was calculated as follows.

Tumor volume (mm) ³ ) = (tumor long diameter× (tumor short diameter) ² )×1/2

TABLE 6

The radioactive complexes produced as described in example 1 and comparative example 1 were each administered in the tail vein at a dose of 15 kBq/dose only (50 μg/dose as trastuzumab). As the control group, a group to which trastuzumab was administered in the same amount as each of the radioactive complexes (antibody control group) and a solvent group to which a preservation buffer was administered were set. The number of groups was 6, and the general state was observed over time up to 38 days after administration, and the body weight and tumor volume were measured. The time-dependent changes in tumor volume are shown in FIG. 1 and the time-dependent changes in body weight are shown in FIG. 2.

The group to which the radioactive complex manufactured as described in example 1 and comparative example 1 was administered showed a significant difference in antitumor effect (P < 0.01) at 38 days after administration compared with 2 control groups (antibody control group, solvent group). In the determination of the significant difference, tukey test was performed using statistical analysis software Stat Preclinica (manufactured by Takumi Information Technology corporation). On the other hand, no significant difference in antitumor effect was observed between the groups to which each of the radioactive complexes was administered. In addition, each group showed no significant change in the general state, and no significant signs of toxicity such as weight loss were observed.

Example 4]Using ¹⁷⁷ Lu-labeled DOTAGA-DBCO for producing trastuzumab complex

(1. Antibody modification step)

The procedure was carried out in the same manner as described in the antibody modification step in example 1.

(2. Complex formation step)

The method for producing DOTAGA-DBCO was carried out in the same manner as in example 1. The chelating agent was dispersed in 0.156mol/L sodium acetate buffer (pH 5.5) as a solvent to prepare a dispersion containing 0.45mmol/L chelating agent. Mixing 0.02mL of the dispersion, 0.02mL of 0.156mol/L sodium acetate buffer (pH 5.5) in which 0.225mmol/L gentisic acid was dissolved, and 25.8MBq (calculated from the decay of radioactivity at the time of the test day) as a radioactive metal source ¹⁷⁷ The reaction solution obtained by the solution of Lu ions (0.04 mol/L hydrochloric acid aqueous solution, radioactivity concentration: 516MBq/mL, preparation by POLATOM, liquid amount: 0.05 mL) was reacted under heating to obtain ¹⁷⁷ Lu complex solution. The molar ratio of chelating agent to radioactive metal ion is chelating agent: ¹⁷⁷ lu ion = about 236:1, the heating temperature of the reaction solution was 70℃and the heating time was 5 minutes.

The obtained ¹⁷⁷ The RCP measurement of Lu complex was performed in the same manner as the RCP measurement of radioactive complex of example 1. As a result of this, the product, ¹⁷⁷ The RCP of Lu complex was 99%. The obtained ¹⁷⁷ The Lu complex solution was directly used in the labeling procedure.

(3. Marking step)

Subjecting the crude product obtained in the step (2) to a purification step ¹⁷⁷ The solution of Lu complex and the solution containing the peptide-modified antibody (monovalent antibody) obtained in the step (1) were added to 0.1mol/L histidine buffer (pH 6.0) containing 0.1mol/L arginine, respectively, and click reaction was performed at 37℃for 120 minutes, thereby obtaining ¹⁷⁷ The Lu complex labels the antibody. ¹⁷⁷ The amount of Lu complex and the amount of peptide-modified antibody (monovalent antibody) were 8.4nmol and 10nmol, respectively, and the molar ratio of dbco groups to azide groups was about 1:1.2. without purification ¹⁷⁷ The reaction rate (%) of the Lu complex-labeled antibody is shown in table 7 below.

In addition, ultrafiltration was performed in the same manner as in example 1After purification by a device ¹⁷⁷ The RCP and RCY of Lu complex labeled antibodies are shown in table 7 below.

¹⁷⁷ The method for measuring RCP and RCY of the Lu complex labeled antibody was carried out in the same manner as in example 1.

TABLE 7

Comparative example 4]Using ¹⁷⁷ Lu-labeled DOTA-DBCO for manufacturing trastuzumab complex

The procedure of example 4 was followed, except that DOTAGA-DBCO was changed to DOTA-DBCO. The results are shown in Table 8.

TABLE 8

Example 5]Using ⁹⁰ Y-labeled DOTAGA-DBCO for preparing trastuzumab complex

(1. Antibody modification step)

(2. Complex formation step)

The method for producing DOTAGA-DBCO was carried out in the same manner as in example 1. The chelating agent was dispersed in 0.156mol/L sodium acetate buffer (pH 5.5) as a solvent to prepare a dispersion containing 0.3mmol/L chelating agent. Mixing 0.03mL of the dispersion, 0.03mL of 0.156mol/L sodium acetate buffer (pH 5.5) in which 0.15mol/L gentisic acid was dissolved, and 113 to 118MBq (calculated from the attenuation of radioactive energy at the time of the test day) as a radioactive metal source ⁹⁰ Y ion solution (0.04 mol/L hydrochloric acid aqueous solution, radioactivity concentration of 3786-3943 MBq/mL, from Eckert)&The reaction mixture obtained by Ziegler-Natta preparation, the liquid amount of which is 0.03 mL) was reacted under heating to obtain ⁹⁰ Y complex solution. The molar ratio of chelating agent to radioactive metal ion is chelating agent: ⁹⁰ y ion=69 to ultra72:1, the heating temperature of the reaction solution was 70℃and the heating time was 5 minutes.

The obtained ⁹⁰ The RCP measurement of the Y complex was performed in the same manner as the RCP measurement of example 1. As a result of this, the product, ⁹⁰ The RCP of the Y complex was 99%. The obtained ⁹⁰ The Y complex solution was used directly in the labeling procedure.

(3. Marking step)

Subjecting the crude product obtained in the step (2) to a purification step ⁹⁰ The solution of the Y complex and the solution containing the peptide-modified antibody (monovalent antibody) obtained in the step (1) were added to 0.1mol/L histidine buffer (pH 6.0) containing 0.1mol/L arginine, and a click reaction was performed at 37℃for 120 minutes, thereby obtaining ⁹⁰ The Y complex labels the antibody. ⁹⁰ The amount of the Y complex and the amount of the peptide-modified antibody (monovalent antibody) were 9nmol and 10nmol, respectively, and the molar ratio of dbco groups to azide groups was about 1:1.1. without purification ⁹⁰ The reaction rate (%) of the Y complex-labeled antibody is shown in table 9 below.

In addition, the mixture was purified by using an ultrafiltration filter in the same manner as in example 1 ⁹⁰ The RCP and RCY of the Y complex labeled antibodies are shown in table 9 below.

⁹⁰ The method for measuring RCP and RCY of the Y complex labeled antibody was carried out in the same manner as in example 1.

TABLE 9

Comparative example 5]Using ⁹⁰ Y-labeled DOTA-DBCO for preparing trastuzumab complex

The procedure of example 5 was followed, except that DOTAGA-DBCO was changed to DOTA-DBCO. The results are shown in Table 10.

TABLE 10

EXAMPLE 6 storage stability of the radioactive Complex produced according to the description of example 4, example 5, comparative example 4 or comparative example 5 (preparation procedure)

A portion of the radioactive complex produced according to the description of example 4, example 5, comparative example 4 or comparative example 5 was extracted into a 0.5mL microcentrifuge tube (manufactured by LoBind, eppendorf Co.) and diluted with a preservation buffer (a mixture of 42g/L trehalose hydrate, 0.47 g/L-histidine hydrochloride hydrate, 0.30 g/L-histidine and 85mg/L polysorbate 20).

[ evaluation 3] stability evaluation

Each of the radioactive complexes obtained in example 6 was stored at room temperature (20.6 to 21.8 ℃) for 2 weeks, and RCP and antigen binding activity were evaluated at each time point (at 0 day, 1 day, 7 days and/or 14 days). The half-life corresponds to about 1 half-life in the case where the radionuclide is Lu-177, about 2.5 half-lives in the case where the radionuclide is Y-90, and about 2 half-lives in the case where the radionuclide is Lu-177, and about 5 half-lives in the case where the radionuclide is Y-90, 7 days from the end of the production, and 14 days from the end of the production.

[ evaluation 3-1] radiochemical purity

RCP was analyzed by TLC. The conditions for TLC were the same as those used in the investigation of the reaction rate in example 1. The results are shown in Table 11.

TABLE 11

The radioactive complex without thiourea bond produced as described in example 4 or 5 was maintained at 95% or more in terms of RCP for 7 days after completion of production. The radioactive complex without thiourea bond produced according to the description of example 4 or 5 was maintained at 90% or more in terms of RCP even when it was stored for 14 days after the end of production.

The radioactive compound produced according to the description of comparative example 4 was less than 85% in terms of RCP when stored at room temperature for 7 days after completion of production, and the radioactive compound produced according to the description of comparative example 5 was less than 75% in terms of RCP when stored at room temperature for 7 days after completion of production. The radioactive compound produced according to the description of comparative example 4 was less than 60% in terms of RCP when it was stored at room temperature for 14 days after the completion of production, and the radioactive compound produced according to the description of comparative example 5 was less than 65% in terms of RCP when it was stored at room temperature for 14 days after the completion of production.

[ evaluation of 3-2] antigen binding Activity

Antigen binding activity was confirmed by ARG in vitro (only at days 0 and 14). The evaluation method was performed in the same manner as described in evaluation 1 to 3. The results are shown in FIGS. 4 and 5.

The radioactive complexes produced as described in example 4, example 5, comparative example 4 or comparative example 5 all confirmed the binding activity to HER2 at the final storage point (14 days). The radioactive complexes manufactured according to the descriptions of example 4, example 5, comparative example 4 or comparative example 5 all bound only to SK-OV-3 tumor sections, showing HER2 selective binding. At the final end of storage (14 days), binding selectivity for HER2 was maintained in all samples. The samples using the radioactive complexes manufactured according to the descriptions of examples 4 or 5 bound more radioactivity to SK-OV-3 tumor sections than the samples using the radioactive complexes manufactured according to the descriptions of comparative examples 4 or 5.

[ evaluation 4]

A subcutaneous tumor-bearing model of SK-OV-3 cells was prepared using mice, and it was confirmed that the radioactive complex prepared as described in example 2 was accumulated in tumors.

SK-OV-3 cells purchased from ATCC as HER 2-positive human ovarian cancer strain were suspended in McCoy's 5A medium (gibco), and 5X 10 cells were subcutaneously administered to the flank of 5-week-old BALB/c nu/nu (Charles River, japan) ⁶ Tumor-bearing mice were prepared from individual cells. Tumor volume of about 100 to 400mm was confirmed 4 weeks after tumor-bearing treatment ³ 。

The radioactive complex manufactured as described in example 2 was administered in tail vein at a dose of 5 MBq/only (n=24). Imaging was performed using a small animal PET imaging device (PET/CT Si78, manufactured by Bruker Co.) 60 hours after administration under the conditions shown in the following table.

Fig. 6 shows a representative example of the results of PET imaging. HER2 positive tumors can be depicted by integrating tumors at a higher concentration of radioactivity than other organs.

TABLE 12

Isotope element	89-Zr
		Acquisition time	600 seconds
Energy window	30％(357.7-664.3keV)
		PET image reconstruction	MLEM GUP 32×32 0.25(Iterations：12)
Correction of	Scattering, randomization, decay, partial volume, decay

Example 7] ²²⁵ Drug efficacy comparison of Ac Complex labeled antibodies and ADC Agents

[ evaluation 5 ]]Uses ²²⁵ Evaluation of drug efficacy of Ac Complex-labeled antibody

Subcutaneous tumor-bearing model of SK-OV-3 was prepared using mice, and was prepared as described in example 1A kind of electronic device ²²⁵ The Ac complex labels the anti-tumor effect of antibodies with commercial Antibody Drug Conjugate (ADC) agents. As the ADC reagent, trastuzumab-delutekang (ENHERTU (registered trademark), first co-product, and trastuzumab-maytansine (Kadcyla (registered trademark), middle and outer pharmaceutical products) were used. In addition, trastuzumab (Herceptin (registered trademark), manufactured by Roche corporation) was used as an antibody control.

SK-OV-3 tumor-bearing mice were prepared by the method described in evaluation 2. After treatment of tumor, the tumor volume was confirmed to be 150 to 550mm ³ The individuals fit to the shape of the tumor diameter were randomly grouped. Tumor volumes and body weights of mice in each group at this time are shown in Table 13. The enherrtu (registered trademark) was classified into a low dose administration group and a high dose administration group according to the administration amount, and the amount of antibody administered in the low dose administration group was adjusted to be the same as that in the high dose administration group ²²⁵ The Ac complex labeled antibody group was equivalent, and the high dose administration group was adjusted to administer the clinical dose as an amount of antibody converted to the weight of the mice.

TABLE 13

The radioactive complex produced as described in example 1 was administered in the tail vein at a dose of 20 kBq/only (20 μg/only as trastuzumab). In addition, herceptin (registered trademark) was administered at a dose of 20 μg/dose, kadcyla (registered trademark) was administered at a dose of 72 μg/dose, and enherrtu (registered trademark) was administered at a dose of 20 μg/dose in the low dose group, and enherrtu (registered trademark) was administered at a dose of 108 μg/dose in the high dose group. The number of groups was 4, and the general state was observed over time up to 35 days after administration, and the body weight and tumor volume were measured. The change in tumor volume of each group of mice at this time is shown in fig. 7.

A significant difference (P < 0.05) was observed in the antitumor effect of the radioactive complex-administered group at 35 days after administration compared to the enherrtu (registered trademark) low dose group. In the test for significant differences, a Tukey test was performed using statistical analysis software Stat Preclinica. Further, at 35 days after administration, a tendency of enhancement of the antitumor effect was confirmed in the radioactive complex administration group as compared with the ADC agent administration group other than the ENHERTUs (registered trademark) low dose group, but no significant difference was observed. No significant change in general status was observed for each group, and no significant weight loss was observed.

EXAMPLE 8 comparison of the efficacy of the respective doses of the radioactive Complex

[ evaluation 6] evaluation of efficacy Using a radioactive Complex

A subcutaneous tumor-bearing model of SK-OV-3 was prepared using mice, and the antitumor effects of the radioactive complex prepared as described in example 1 were compared with those of a commercially available ADC drug. As the ADC reagent, trastuzumab-delutetratecan (ENHERTU (registered trademark), first co-product).

SK-OV-3 tumor-bearing mice were prepared by the method described in evaluation 2. The tumor volume was confirmed to be 100 to 300mm 3 weeks after the tumor-bearing treatment ³ The individuals fit to the shape of the tumor diameter were randomly grouped. Tumor volumes and body weights of the mice at this time are shown in table 14.

TABLE 14

The radioactive complex produced as described in example 1 was administered in tail vein at a dose of 20kBq/21g in the high-radioactivity administration group, at a dose of 10kBq/21g in the medium-radioactivity administration group, and at a dose of 5kBq/21g (each group was 3.57mg/kg as trastuzumab) in the low-radioactivity administration group. The ADC agent was administered tail-vein at a medium dose of 10 mg/kg. In addition, a group (antibody control group) to which trastuzumab was administered in the same amount as the radioactive complex administration group (3.57 mg/kg in terms of trastuzumab) and a solvent group to which a preservation buffer was administered were set. The number of groups was 6, and the general state was observed over time up to 46 days after administration, and the body weight and tumor volume were measured. The change in tumor volume of each group of mice at this time is shown in fig. 8. In addition, anatomical examination was performed on the last day of observation, heart, lung, spleen, liver and kidney were collected, and their weights were measured.

Significant differences (P < 0.01) in anti-tumor effect were observed for each of the radioactive complex-dosed groups compared to the antibody control group and the solvent group at 46 days post-dosing. In addition, the tendency that the antitumor effect was enhanced depending on the administered radioactivity was confirmed, suggesting that the antitumor effect was enhanced depending on the administered radioactivity. In the test for significant differences, a Tukey test was performed using statistical analysis software Stat Preclinica. Further, at 46 days of administration, no significant difference was observed in the antitumor effect between the radioactive complex high-radioactivity group and the radioactive complex medium-radioactivity group, as compared with the ADC agent administration group, suggesting that the antitumor effect was equivalent. No significant change in general status was observed for each group, and no significant weight loss was observed. In addition, no significant difference was observed in the weight of the organs collected by anatomical examination on the last day of observation.

Example 9]Using ²²⁵ Ac-labeled DOTAGA-DBCO for producing pertuzumab complex

(1. Antibody modification step)

A peptide comprising the amino acid residue represented by the above formula (P3) was obtained in the same manner as in example 1.

The peptide and pertuzumab (Perjeta (registered trademark), extra and Extra pharmaceutical Co., ltd.) were mixed in 0.02mol/L sodium acetate buffer (pH 6.0), and the resulting mixture was allowed to react at room temperature for 60 minutes to obtain a solution containing the peptide-modified antibody. The peptide-modified antibody is obtained by site-specifically modifying the Fc region of an antibody with the above-mentioned peptide.

Then, the IgG-BP column was subjected to liquid chromatography to obtain an antibody composition comprising a relatively large amount of unlabeled antibody and monovalent antibody. The concentration of the monovalent antibody contained in the recovered fraction was adjusted to 14.6mg/mL by using a preservation buffer (41 g/L sucrose, 3.1g/L histidine, 0.66g/L glacial acetic acid mixture) (pH 6.0). The labeling step described below is performed by supplying a solution containing a relatively large amount of unlabeled antibody and monovalent antibody.

(2. Complex formation step)

DOTAGA-DBCO was produced in the same manner as in example 1. The chelating agent was dispersed in 0.156mol/L sodium acetate buffer (pH 5.5) as a solvent to prepare a dispersion containing 0.3mmol/L chelating agent. Mixing 0.02835mL of the dispersion with 3.25MBq (calculated from the decay of the radiant energy at the test day) of the metal source ²²⁵ The reaction solution obtained by the reaction of the Ac ion solution (0.1 mol/L hydrochloric acid aqueous solution, radioactivity concentration of 259MBq/mL, prepared by Rosatom State Atomic Energy Corporation, liquid amount of 0.0126 mL) was heated to obtain ²²⁵ Ac complex solution. The molar ratio of chelating agent to radioactive metal ion at this time is chelating agent: ²²⁵ ac ion = about 1270:1, the heating conditions of the reaction solution were 70℃and the heating time was 30 minutes.

The obtained product was measured in the same manner as in example 1 ²²⁵ RCP of Ac complex. As a result of this, the product, ²²⁵ the RCP of the Ac complex was 67%. The obtained ²²⁵ The Ac complex solution was directly used in the labeling procedure.

(3. Marking step)

Subjecting the crude product obtained in the step (2) to a purification step ²²⁵ The Ac complex solution was mixed with the solution containing the peptide-modified antibody (monovalent antibody) obtained in the step (1), and the click reaction was carried out at 37℃for 2 hours, thereby obtaining ²²⁵ The Ac complex labels the antibody. The molar ratio of DBCO groups to azide groups was about 1:1.3. without purification ²²⁵ The reaction rates of the Ac complex-labeled antibodies are shown in Table 15 below.

Purifying with ultrafiltration filter (manufactured by Merck Co., ltd.: UFC 803096) and reacting at 37deg.C for 2 hours ²²⁵ The Ac complex labels the solution of the antibody. Purified ²²⁵ The RCP and RCY of the Ac complex labeled antibodies are shown in table 15 below. It should be noted that the number of the substrates, ²²⁵ the RCP and RCY of the Ac complex-labeled antibody were calculated from the radioactivity obtained by the same method as in example 1.

TABLE 15

EXAMPLE 10 formulation procedure

A portion of each of the radioactive complexes produced as described in example 9 was extracted into a 0.5mL microcentrifuge tube (manufactured by LoBind, eppendorf Co.) and diluted with a preservation buffer (41 g/L sucrose, 3.1 g/L-histidine, 0.66g/L glacial acetic acid, and 0.2g/L polysorbate 20 mixture).

[ evaluation 7] stability evaluation

The radioactive complex obtained in example 9 was stored at room temperature (24.5 to 25.5 ℃) for 2 weeks, and the proportions of RCP and aggregates were evaluated at each time point (0 day, 1 day, 7 days and 14 days).

[ evaluation 7-1] RCP

The RCP was calculated from the TLC analysis results. The conditions for TLC were the same as those used in the evaluation of the reaction rate in example 1. The results are shown in Table 16.

TABLE 16

The radioactive complex produced as described in example 9 was maintained at 98% or more in terms of RCP when stored at room temperature for 7 days after completion of production. When the resin composition is stored at room temperature for 14 days after completion of production, 97% or more of the resin composition is maintained as RCP.

[ evaluation 7-2] proportion of aggregates

The proportion of aggregates was confirmed by SEC in the same manner as described in example 1. The proportions of the components at each evaluation day are shown in Table 17 when they were stored for 14 days after the completion of production. The proportion of aggregates in the case of storage for 14 days after completion of production was 1.66%.

TABLE 17

	Proportion of main peak (%)	Peak ratio of aggregate (%)
			Radioactive complex (example 9), 0 day after manufacture	99.77	0.23
Radioactive complexes (example 9), 1 day after manufacture	98.68	0.25
			Radioactive complex (example 9), 7 days after manufacture	93.28	0.47
Radioactive complexes (example 9), 14 days after manufacture	89.51	1.66

The present application is based on Japanese patent application 2020-174840 (application date: 10/16/2020), japanese patent application 2020-215740 (application date: 24/2020), and Japanese patent application 2021-024688 (application date: 18/2021/2/2020), the contents of which are all incorporated herein.

Claims

1. A complex of an anti-HER 2 antibody site-specifically modified with a peptide and a chelating agent,

the radionuclides are sequestered in the chelating agent,

the peptide is linked to the chelating agent via a linker L, which does not contain thiourea bonds.

2. The complex of claim 1, wherein the chelating agent is dotga, i.e., α - (2-carboxyethyl) -1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid.

3. The complex according to claim 1, wherein the peptide has an amino acid sequence of 13 to 17 amino acid residues represented by the following formula:

(Xa)-Xaa1-(Xb)-Xaa2-(Xc)-Xaa3-(Xd)···(i)

x is an amino acid residue having no one of a thiol group and a haloacetyl group in a side chain,

xaa1 and Xaa3 each independently represent:

xaa1 and Xaa3 are joined to form a ring structure,

4. A complex according to any one of claims 1 to 3 wherein the radionuclide is Ac-225, Y-90, lu-177 or Zr-89.

5. The complex according to any one of claims 1 to 4, wherein the linker L comprises the formula (10 a), the formula (10 b) or the formula (10 c):

[ chemical formula 1]

6. The radioactive complex of claim 5, wherein a polyethylene glycol group is contained between a site of attachment to the peptide and the peptide.

7. The radioactive complex according to any one of claims 1 to 6, wherein the anti-HER 2 antibody is complexed by a click reaction with a radioactive metal complex of dotga-DBCO represented by the following formula, the anti-HER 2 antibody being site-specifically modified with the peptide having an azide group introduced at the N-terminus:

[ chemical formula 2]

8. The complex of any one of claims 1-7, wherein the anti-HER 2 antibody is trastuzumab or pertuzumab.

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

10. The radiopharmaceutical of claim 9 for use in the radiation therapy of cancer.

11. The radiopharmaceutical of claim 9 for use in diagnosis of cancer.

12. The radiopharmaceutical of claim 11 for use in combination with an intra-radiotherapy for cancer using the radiopharmaceutical of claim 10.

13. A radiopharmaceutical comprising, as an active ingredient, a complex of a chelator chelated with a radiometal nuclides and an anti-HER 2 antibody,

and satisfies the following condition (1) or (2):

14. A radiopharmaceutical comprising, as an active ingredient, a complex of a chelator chelated with a radiometal nuclides and an anti-HER 2 antibody,