Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
  • A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
  • A61K51/0478—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group complexes from non-cyclic ligands, e.g. EDTA, MAG3
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
  • A61K51/1096—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies radioimmunotoxins, i.e. conjugates being structurally as defined in A61K51/1093, and including a radioactive nucleus for use in radiotherapeutic applications
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2887—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates to a method for producing a radioactive metal-labeled antibody.
• the antibody is used as a reagent for detecting the target molecule, a diagnostic agent, or a drug for treating a disease by utilizing the specificity of the antibody for the target molecule. For the purpose of further improving the detection performance and the therapeutic effect, studies on antibodies to which radionuclides and drugs are bound are underway.
• Patent Document 1 describes a peptide containing an amino acid sequence consisting of 13 to 17 amino acid residues and capable of binding to a human antibody.
• the document also states that the peptide can be modified by a labeling substance such as a complex containing a radioisotope or a drug such as an anticancer drug, and that a complex of the peptide and an antibody can be formed.
• a labeling substance such as a complex containing a radioisotope or a drug such as an anticancer drug
• Patent Document 2 describes a complex of an antibody and a peptide modified with a DTPA complex of 111 In, which is a radioactive metal. It has also been described that this antibody complex specifically binds to a target molecule expressed in a tumor.
• Patent Documents 1 and 2 have not examined any reaction conditions for solving such problems.
• an object of the present invention is to provide a method for producing a radioactive metal-labeled antibody, which is excellent in labeling efficiency of the radioactive metal with respect to the antibody even under mild reaction conditions.
• the present invention comprises a step of producing a radiometal-labeled antibody by click-reacting a radiometal complex with an antibody site-specifically modified with a peptide.
• the click reaction is performed between the first atomic group of the radioactive metal complex and the second atomic group that is directly or indirectly linked to the peptide. It provides a method for producing a radiometal-labeled antibody, wherein the second atomic group is an atomic group containing an azide group or an atomic group containing trans-cyclooctene.
• the present invention is a radiometal-labeled antibody site-specifically modified with a peptide.
• the radioactive metal complex is directly or indirectly linked to the peptide and has a triazole skeleton-containing structure represented by the following formula (10a) between the peptide and the radioactive metal complex, or the peptide and the said. It provides a radiometal-labeled antibody having a pyridazine skeleton-containing structure between the radiometal complex and the radiometal complex.
• R 1A indicates a binding site with a modified part or a chelating part
• R 2A indicates a binding site with a peptide.
• the present invention has a chelating portion coordinated to a radioactive metal and a modified portion having a first atomic group capable of click reaction, and the first atomic group is represented by either the following formula (1a) or (1b). It provides a chelate linker, which is a represented atomic group.
• R 1 indicates a binding site with a modified part or a chelating part
• R 3 and R 4 indicates a binding site with a modified part or a chelating part.
• the other indicates a hydrogen atom, a methyl group, a phenyl group or a pyridyl group.
• the present invention is a peptide-modified antibody that is site-specifically modified with a peptide containing an amino acid sequence consisting of 13 to 17 amino acid residues represented by the following formula (i).
• Xa, Xb, Xc and Xd represent a continuous X, a continuous b X, a continuous c X, and a continuous d X, respectively.
• X is an amino acid residue that has neither a thiol group nor a haloacetyl group in the side chain.
• a, b, c and d are each independently an integer of 1 or more and 5 or less, and satisfy a + b + c + d ⁇ 14.
• Xaa1 and Xaa3 are independent of each other Represents an amino acid residue derived from an amino acid having a thiol group in the side chain, and is bound via a disulfide bond or the sulfide group is bound via a linker.
• One represents an amino acid residue derived from an amino acid having a thiol group in the side chain, and the other represents an amino acid residue derived from an amino acid having a haloacetyl group in the side chain, and is bound via a thioether bond.
• Xaa2 is a lysine residue, an arginine residue, a cysteine residue, an aspartic acid residue, a glutamate residue, 2-aminosveric acid, or a diaminopropionic acid.
• R 2 indicates the binding site of the peptide to the N-terminal or C-terminal
• R 5 indicates the binding site of the peptide to the N-terminal or C-terminal.
• the labeling efficiency of radioactive metals with respect to antibodies is excellent even under mild reaction conditions.
• a radioactive metal complex is click-reacted with an antibody site-specifically modified with a peptide (hereinafter, this is also simply referred to as “peptide-modified antibody”) to generate a radioactive metal-labeled antibody. It is equipped with a process (labeling process). Details of the radioactive metal, its complex, and the peptide will be described later.
• the radioactive metal complex and the peptide-modified antibody each have a click-reactive atomic group, and these atomic groups react with each other so that the radioactive metal complex and the peptide-modified antibody can bind to each other. That is, the click reaction in this step is carried out between the first atomic group contained in the radioactive metal complex and the second atomic group directly or indirectly linked to the peptide in the peptide-modified antibody. is there.
• “Directly or indirectly” refers to whether or not a linker structure, which will be described later, is formed between the second atomic group and the peptide. "Directly” means having no linker structure, and more specifically, that the second atomic group is attached to the N-terminus or C-terminus of the peptide without going through the linker structure. Further, “indirectly” means having a linker structure, and more specifically, it means that the second atomic group is bound to the N-terminal or C-terminal of the peptide via the linker structure. .. In either “directly” or “indirectly” embodiment, the second atomic group is preferably attached to the N-terminal side of the peptide.
• the above-mentioned linker structure is represented by the following formula (S1). **-((L 1 ) m- Z) k- L 2- AG 2 ... (S1) (In the formula, ** indicates the binding site of the peptide to the N-terminal or C-terminal, and indicates the binding site.
• L 1 is a polyethylene glycol (PEG) linker portion, and is m is an integer of 1 or more and 50 or less
• Z is a second linker portion that binds (L 1 ) m and L 2.
• k is 0 or 1 and L 2 is the second PEG linker moiety and AG 2 is the second atomic group.
• the structure of Z is not particularly limited as long as it is a linker structure that binds (L 1 ) m and L 2 to each other, and includes, for example, an amino acid sequence consisting of amino acid residues of 1 or more and 5 or less. be able to.
• the amino acid sequence contained in Z preferably contains a cysteine residue, and is bound to L 2 via a thioether group formed by the bond between the thiol group and the maleimide group of the cysteine residue. More preferred.
• PEG linker portion constituting the L 2 are preferably has a structure as shown in formula (P1).
• n is an integer, preferably 1 or more and 50 or less, more preferably 1 or more and 20 or less, and further preferably 2 or more and 10 or less.
• One end of the structure of the PEG linker portion may be modified by a structure derived from a commercially available PEGylation reagent or a structure derived from a reagent usually used for PEGylation, and is not particularly limited, but for example, diglycol. Examples are structures derived from acids or derivatives thereof, maleimides or derivatives thereof.
• an appropriate combination of atomic groups capable of click reaction is selected according to the type of click reaction.
• a combination of alkyne and azide, 1,2,4,5-tetrazine and alkene are used. Examples include the combination of.
• the first atomic group may have one of the above atomic groups
• the second atomic group may have an atomic group that is a combination of the first atomic group. From the viewpoint of achieving both the stability of the radioactive metal complex and the peptide-modified antibody and the improvement of their binding efficiency, the first atomic group is an alkyne and the second atomic group is an azide, or the first atomic group is an azide.
• 1,2,4,5-tetrazine and the second atomic group is an alkene.
• Specific examples of the click reaction by such a combination of atomic groups include a Husgen cycloaddition reaction, an inverse electron-requested Diels-Alder reaction, and the like.
• the combination of click-reactive atomic groups include an atomic group containing dibenzylcyclooctin (DBCO) as an alkyne of the first atomic group (formula (1a)) and a second atomic group, as shown in the following formula.
• DBCO dibenzylcyclooctin
• a combination with an atomic group containing an azide group (formula (2a)) as the azide of the atomic group, or an atomic group containing 1,2,4,5-tetrazine as the first atomic group (formula (1b)) and the first Examples of the alkene of the two atomic groups include a combination with an atomic group containing trans-cyclooctene (TCO) (formula (2b)).
• R 1 indicates a binding site with a modified part or a chelating part
• R 2 indicates a binding site with a peptide in a peptide-modified antibody.
• R 3 and R 4 shows a binding site with another structure, and the other shows a hydrogen atom, a methyl group, a phenyl group or a pyridyl group, and in formula (2b), R 5 Indicates the binding site with other structures.
• one of the radioactive metal complex and the peptide-modified antibody is added to a reaction vessel containing a solvent regardless of the order of addition thereof. Then, the other may be added and reacted, or the other may be added and reacted in a dispersion in which one of the radioactive metal complex and the peptide-modified antibody is dispersed in a solvent. Alternatively, these may be added at the same time to a reaction vessel containing a solvent to cause a reaction.
• a solvent containing water can be used, and for example, water, physiological saline, or sodium acetate buffer, ammonium acetate buffer, phosphate buffer, phosphate buffered saline, etc.
• Trishydroxymethylaminomethane buffer hereinafter, simply referred to as "Tris buffer”
• HEPES buffer 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid buffer
• a buffer solution such as a methylammonium acetate buffer solution can be used.
• a buffer solution the lower limit of the pH at 25 ° C.
• the upper limit of the reaction temperature of the click reaction in this step is preferably 120 ° C. or lower, preferably 90 ° C. or lower. Is more preferable, 50 ° C. or lower is further preferable, and even more preferably 40 ° C. or lower.
• the lower limit of the reaction temperature is not particularly limited as long as it can be click-reacted, but is preferably 10 ° C. or higher, more preferably 15 ° C. or higher, further preferably 20 ° C. or higher, further preferably 30 ° C. or higher, and 35. °C or higher is particularly preferable.
• the reaction time of the click reaction is preferably 5 minutes or more, more preferably 10 minutes or more, further preferably 20 minutes or more, further preferably 30 minutes or more, and more preferably 60 minutes, provided that the reaction temperature is the above-mentioned reaction temperature. Minutes or more are particularly preferable, the upper limit is preferably 36 hours or less, more preferably 24 hours or less, further preferably 20 hours or less, particularly preferably 15 hours or less, and the preferred range is 5 minutes or more and 24 hours or less, further preferable range. Is 10 minutes or more and 20 hours or less.
• the amount of the reaction solution is not particularly limited, but from the viewpoint of practicality in the production process, the lower limit at the start of this step is preferably 0.01 mL or more, more preferably 0.1 mL or more, still more preferably 1 mL or more.
• the upper limit is preferably 1000 mL or less, more preferably 100 mL or less, still more preferably 10 mL or less, and for example, 0.1 mL or more and 10 mL or less.
• the concentrations of the radioactive metal complex and the peptide-modified antibody in the reaction solution are independently, at the start of this step, the lower limit is preferably 0.01 ⁇ mol / L or more, more preferably 0.1 ⁇ mol / L or more.
• the upper limit is preferably 10,000 ⁇ mol / L or less, more preferably 1000 ⁇ mol / L or less, still more preferably 100 ⁇ mol / L or less, for example, 1 ⁇ mol / L or more and 100 ⁇ mol / L or less.
• the yield of the target radioactive metal-labeled antibody it is preferable from the viewpoint of the yield of the target radioactive metal-labeled antibody.
• the obtained radioactive metal-labeled antibody may be used as it is, or may be purified by using a filtration filter, a membrane filter, a column packed with various fillers, chromatography or the like.
• the radioactive metal complex and the peptide-modified antibody can be bound to each other under mild reaction conditions to obtain a high yield of the antibody labeled with the radioactive metal. Further, since the obtained labeled antibody can be specifically labeled at a site that does not inhibit the antigen specificity of the antibody, the antigen specificity of the antibody itself is maintained.
• the binding reaction between the complex and the antibody can proceed sufficiently and in a short time without performing heat treatment for promoting the binding reaction between the radioactive metal complex and the peptide-modified antibody, and thus positrons. Even when a radioactive metal having a short half-life such as a released nuclei is used, a labeled antibody having high radiochemical purity and radiochemical yield can be obtained in a short time.
• the production method of the present invention is a step of reacting a ligand with a radioactive metal to form a radioactive metal complex from the viewpoint that the labeled radioactive metal can be used without particular limitation according to the use and purpose of the labeled antibody ( It is preferable to provide a complex forming step) before the labeling step.
• the ligand used in this step preferably has a first atomic group.
• the radioactive metal in this step is preferably used in the form of an ionizable radioactive metal compound, and more preferably in the form of a radioactive metal ion (hereinafter, these aspects are collectively referred to) from the viewpoint of increasing the complex formation efficiency. It is also called "radioactive metal source").
• the complex formation may not proceed well under non-heating conditions.
• the complex formation may not proceed well. Complex formation can proceed efficiently without depending on the combination of the ligand and the radioactive metal.
• the addition order of the ligand and the radioactive metal source does not matter, for example, one of the ligand and the radioactive metal source is added to the reaction vessel containing the solvent. Then, the other may be added and reacted, or the other may be added and reacted in a dispersion in which one of the ligand and the radioactive metal source is dispersed in a solvent. Alternatively, these may be added at the same time to a reaction vessel containing a solvent to cause a reaction.
• the reaction conditions in the complex formation step can be, for example, the following conditions.
• the solvent used in this step include water, physiological saline, sodium acetate buffer, ammonium acetate buffer, phosphate buffer, phosphate buffer physiological saline, Tris buffer, HEEPS buffer, or tetramethyl.
• a buffer solution such as an ammonium acetate buffer solution, or a water-soluble organic solvent such as alcohol having 1 or more and 5 or less carbon atoms, acetonitrile, N, N-dimethylformiamide, tetrahydrofuran, dimethylsulfoxide and acetone, or a mixed solvent thereof. be able to.
• the reaction temperature may be, for example, room temperature (25 ° C.) or heating conditions, but the upper limit is 120 ° C. or lower from the viewpoint of suppressing the decomposition of the ligand and improving the complex formation efficiency.
• the temperature is preferably 90 ° C. or lower, more preferably 50 ° C. or lower, and even more preferably 40 ° C. or lower.
• the lower limit is not particularly limited as long as the temperature allows a click reaction, but is preferably 0 ° C. or higher, more preferably 10 ° C. or higher, further preferably 15 ° C. or higher, further preferably 20 ° C. or higher, and more preferably 30 ° C. or higher. Even more preferably, 35 ° C. or higher is particularly preferable.
• the lower limit of the reaction time is preferably 5 minutes or more, more preferably 10 minutes or more, further preferably 20 minutes or more, further preferably 30 minutes or more, and particularly preferably 60 minutes, provided that the reaction temperature is the above-mentioned reaction temperature. It is minutes or more, and the upper limit is preferably 300 minutes or less, more preferably 150 minutes or less, further preferably 120 minutes or less, particularly preferably 60 minutes or less, preferably 10 minutes or more and 150 minutes or less, still more preferably 30 minutes. Minutes or more and 60 minutes or less.
• radioactive metal source in the complex forming step for example, a solution in which radioactive metal ions are dispersed or dissolved in a solvent mainly composed of water can be used.
• the amount of the reaction solution in this step is not particularly limited, but from the viewpoint of practicality in the manufacturing process, the lower limit at the start of this step is preferably 0.01 mL or more, more preferably 0.1 mL or more, still more preferably 1 mL.
• the upper limit is preferably 1000 mL or less, more preferably 100 mL or less, still more preferably 10 mL or less, and for example, 0.01 mL or more and 100 mL or less.
• the concentrations of the ligand and the radioactive metal ion in the reaction solution are independently, at the start of this step, the lower limit is preferably 0.01 ⁇ mol / L or more, more preferably 0.1 ⁇ mol / L or more, still more preferable.
• Is 1 ⁇ mol / L or more and the upper limit is preferably 10000 ⁇ mol / L or less, more preferably 1000 ⁇ mol / L or less, still more preferably 100 ⁇ mol / L or less, for example, 1 ⁇ mol / L or more and 100 ⁇ mol / L or less. It is preferable from the viewpoint of the yield of the target radioactive metal complex.
• the molar ratio of ligand and radiometal ion varies depending on the type of ligand and radiometal ion used, but the lower limit of the ligand / radiometal ion is preferably 10/1 or more, more preferably 100/1 or more, and 200.
• the upper limit is preferably 10000/1 or less, more preferably 9000/1 or less, further preferably 8000/1 or less, and more. It is more preferably 7000/1 or less, preferably 200/1 or more and 10000/1 or less, and particularly preferably 500/1 or more and 7000/1 or less.
• the obtained radioactive metal complex may be used as it is, or may be purified by using a filtration filter, a membrane filter, a column filled with various fillers, chromatography or the like.
• a filtration filter a membrane filter, a column filled with various fillers, chromatography or the like.
• complex formation proceeds well and the yield of the product is high. It is advantageous in that the complex containing the metal nuclide can be used in the subsequent steps in an unpurified state.
• the ligand is used at the site where the radioactive metal ion is coordinated. It is preferable to have a certain chelating part and a modified part bonded to the first atomic group.
• the modified portion (indicated by Rm in the formula (3a)) is bound to the above-mentioned chelating portion (indicated by Ch in the formula (3a)). And the above-mentioned first atomic group (indicated by AG in the formula (3a)) is bonded.
• Rm is a linear or branched chain, substituted or unsubstituted, and an atomic group having a total carbon number of 10 or more and 50 or less.
• the modified portion Rm is not particularly limited in the bonding mode with the chelating portion Ch as long as the complex can be formed between the ligand and the radioactive metal ion, but from the viewpoint of efficiently forming the complex between the ligand and the radioactive metal ion, the modified portion Rm is not particularly limited. It is preferable that the modified portion Rm and the chelated portion Ch form a thiourea bond and bond with each other, or the modified portion Rm and the chelated portion Ch form an amide bond and bond with each other. Further, from the viewpoint of increasing the labeling efficiency of the radioactive metal complex and the antibody, the modified portion Rm binds to R 1 and R 3 or R 4 represented by the above formulas (1a) and (1b) in the first atomic group. It is also preferable that
• the modified portion has a structure represented by the following formula (P2) bonded to the structure represented by Rm in the formula (3a).
• the structure is derived from ethylene glycol, and in the formula (P2), r is preferably an integer of 2 or more and 50 or less, and more preferably an integer of 2 or more and 30 or less.
• the chelate portion is not particularly limited as long as it has a site in which the radioactive metal is coordinated in the structure, but is not particularly limited, but CB-TE2A (1,4,8,11-Tetraazabiciclo [6.6.2] hexadecane-4].
• R 11 , R 13 and R 14 are independently ⁇ (CH 2 ) p COOH, ⁇ (CH 2 ) p C 5 H 5 N, ⁇ (CH 2 ) p PO 3 H, respectively.
• the other is a group bonded to the above-mentioned modified portion, and p is an integer of 0 or more and 3 or less.
• R 21 , R 22 , R 23 and R 24 are independently carboxyl groups or carboxylalkyl groups having 2 or 3 carbon atoms, but R 21 , R 22 , R 23 or R 23 or Any one group of R 24 is a group bonded to the above-mentioned modified portion.
• R 31 , R 32 , R 33 and R 34 each independently have a hydrogen atom and a carbon atom of 2 or more and 10 or less, and may contain a nitrogen atom or an oxygen atom. It is a group composed of atomic groups, and R 35 is a group bonded to the above-mentioned modified portion.
• one of R 41 or R 42 has a hydrogen atom and a carbon atom of 5 or more and 20 or less, and is a group consisting of an atomic group containing one or more selected from a nitrogen atom, an oxygen atom and a sulfur atom. And the other is a group bonded to the above-mentioned modifier.
• R 51 , R 52 , R 53 , R 54 and R 55 are independently carboxyl groups or carboxylalkyl groups having 2 or 3 carbon atoms, except that R The group of any one of 51 , R 52 , R 53 , R 54 or R 55 is a group bonded to the above-mentioned modifier.
• R 61 , R 62 , R 63 , R 64 , R 65 and R 66 are independently carboxyl groups or carboxylalkyl groups having 2 or 3 carbon atoms, and R Reference numeral 67 is a group bonded to the above-mentioned modifier.
• R 71 and R 72 are ⁇ O (CH 2 CH 2 O) n CH 3 (where n is an integer of 1 or more and 5 or less), and are R 73 , R 75 , R 76 and R. 78 is an alkyl group having 1 to 5 carbon atoms independently, and R 74 or R 77 is a hydroxyalkyl group having 1 to 5 carbon atoms, and the other is the modified portion. It is a bonded group.
• R 81 and R 82 are independently alkyl groups having 1 or more and 5 or less carbon atoms, and the ends of the alkyl groups are substituted with pyridyl groups substituted with 1 or more carboxyl groups.
• R 84 are optionally substituted pyridinyl groups
• R 85 and R 86 are independently ⁇ COORa
• Ra is an alkyl group having 1 to 5 carbon atoms.
• R 91 , R 92 , R 93 and R 94 are independently ⁇ OCH 2 COOH, but any one group of R 91 , R 92 , R 93 or R 94 is used.
• R 95 , R 96 , R 97 and R 98 are each independently an alkyl group having 1 or more and 6 or less carbon atoms.
• R 101 , R 102 and R 103 are independently carboxyl groups or carboxylalkyl groups having 2 or 3 carbon atoms, or in formula (J), R. At least one of 101, R 102 and R 103 is a group bonded to the above-mentioned modified portion, and the other group is a carboxyl group or a carboxylalkyl group having 2 or 3 carbon atoms.
• the "group bonded to the modified portion” is a carboxyl group, an amino group, an N-hydroxysuccinimide ester (NHS) group, or 2,6-dioxotetrahydro-2H-pyranyl.
• a structure derived from a group, an isocyanate group, or an isothiocyanate group and a structure in which a modified portion is bonded are shown.
• the binding site between the chelate portion and the modified portion is preferably an amide bond or a thiourea bond as described above, but an amide bond is more preferable from the viewpoint of further increasing the yield.
• the amide bond is, for example, the above formulas (B-1) to (B-2), (G-1), (H-1) to (H-4), (I-1), (J-1) to.
• the amino group, hydroxy group, carboxyl group, or NHS group formed by the reaction of the oxotetrahydro-2H-pyranyl group with the primary amine or shown at the right end in the figure of the compound represented by the formula (K). It is formed by the reaction with the reagent having.
• the thiourea bond is a reaction between the isothiocyanate group of the compound represented by the above formulas (A-2), (A-3), (D-2) or (F-2) and a primary amine or maleimide group. Is formed by.
• the modified portion can be formed by selecting variously from a commercially available reagent containing a primary amine or a commercially available reagent capable of forming an amide bond or a thiourea bond to which a desired primary atomic group is bonded.
• DBCO-amine, DBCO-maleimide, DBCO-PEG-NHS ester, DBCO-PEG-alcohol, DBCO -PEG-amine, DBCO-PEG-maleimide and the like can be selected, but preferably DBCO-amine, DBCO-maleimide, DBCO-PEG-amine, DBCO-PEG-maleimide and the like can be selected.
• the radioactive metal complex is not particularly limited as long as it is formed by reacting a radioactive metal with a ligand having a structure in which an appropriate one is selected from the above-mentioned first atomic group, chelating part and modifying part.
• the radioactive metal complex is preferably formed by reacting a ligand having a structure represented by the following formula (ii) with a radioactive metal. ABC ⁇ ⁇ ⁇ (ii) In formula (ii), A is represented by the following formula (iia).
• Ra, Rb and Rc are independently ⁇ (CH 2 ) p COOH, ⁇ (CH 2 ) p C 5 H 5 N, ⁇ (CH 2 ) p PO 3 H 2 , ⁇ ( CH 2 ) It is a group consisting of p CONH 2 or-(CHCOOH) (CH 2 ) p COOH, p is an integer of 0 or more and 3 or less, and either Rd or Re is a binding site with B ( *) And the other is a hydrogen atom, or-(CH 2 ) p COOH,-(CH 2 ) p C 5 H 5 N,-(CH 2 ) p PO 3 H 2 ,-(CH 2 ) p It is a group consisting of CONH 2 or ⁇ (CHCOOH) (CH 2 ) p COOH, and p is an integer of 0 or more and 3 or less.
• B is represented by the following equation (iib).
• La and Lb are independently binding linkers having at least an amide bond or a thiourea bond and having 1 to 50 carbon atoms, t is an integer of 0 to 30 and s is 0 or 1, * is the binding site with A, and ** is the binding site with C.
• C is either an alkyne derivative represented by the following formula (iic) or a tetrazine derivative represented by the formula (iid).
• X is CHRk-** or N-**
• Rk is an independently hydrogen atom or an alkyl group having 1 or more and 5 or less carbon atoms.
• the Rk moieties may be combined to form a cycloalkyl group, with Rf, Rg, Rh and Ri independently.
• a hydrogen atom, a halogen atom, or an alkyl group having 1 to 5 carbon atoms, Rf and Rg may be combined, or Rh and Ri may be combined to form a hydrocarbon ring, where ** is B.
• ** indicates a binding site with B
• Rj indicates a hydrogen atom, a methyl group, a phenyl group or a pyridyl group.
• the ligand is a DOTA derivative in the above formula (iia) in which Ra to Rd are ⁇ (CH 2 ) p COOH, p is 1, and Re is a binding site with B; or Ra.
• Ra to Rd are ⁇ (CH 2 ) p COOH, p is 1, and Re is a binding site with B; or Ra.
• Rc is ⁇ (CH 2 ) p COOH, p is 1
• Rd is the binding site (*) with B, and either a DO3A derivative or a DOTAG A derivative in which Re is a hydrogen atom is more preferable. ..
• A when A is the DOTA derivative, B is a binding linker having 1 to 50 carbon atoms including a thiourea bond, s is 0 or 1, and s is 1. Is an integer of 0 or more and 30 or less, Lb is a binding linker having 1 or more and 50 or less carbon atoms containing an amide bond or a thiourea bond, and C is an alkin derivative represented by the formula (iic).
• X is N-**
• Y is CHRk
• Rk is a hydrogen atom
• Rf and Rg together form a benzene ring
• Rh and Ri together.
• ** is a bond site with B, a DOTA-PEGt-DBCO derivative; or B is a bond linker having La of 1 to 50 carbon atoms containing a thiourea bond.
• s is 0 or 1
• t is an integer of 0 or more and 30 or less
• Lb is a binding linker having 1 or more and 50 or less carbon atoms including an amide bond or a thiourea bond
• C is.
• DOTA-PEGt-Tz derivative which is a tetrazine derivative represented by the formula (iid) is even more preferable.
• B is a binding linker in which La contains an amide bond or a thiourea bond and has 1 to 50 carbon atoms, s is 0 or 1, and s is 1.
• t is an integer of 0 or more and 30 or less
• Lb is a binding linker having 1 or more and 50 or less carbon atoms containing an amide bond or a thiourea bond
• C is an alkyne derivative represented by the formula (iic).
• X is N-**
• Y is CHRk
• Rk is a hydrogen atom
• Rf and Rg are combined to form a benzene ring
• Rh and Ri A DO3A-PEGt-DBCO derivative, which forms a benzene ring together with each other and where ** is a binding site with B, is even more preferable.
• B is a binding linker having 1 to 50 carbon atoms including an amide bond or a thiourea bond
• s is 0 or 1
• s is 1.
• t is an integer of 0 or more and 30 or less
• Lb is a binding linker having an amide bond or a thiourea bond and having 1 to 50 carbon atoms
• C is represented by the formula (iic). It is an alkyne derivative, and in formula (iic), X is N-**, Y is CHRk, Rk is a hydrogen atom, Rf and Rg are combined to form a benzene ring, and Rh. And Ri together to form a benzene ring, where ** is a binding site with B, a DOTAG A-PEGt-DBCO derivative is even more preferred.
• a specific site of the antibody is specifically modified by the peptide, preferably the Fc region (constant region) of the antibody is specifically modified, and particularly preferably the Fc region of the antibody.
• the lysine residue in is site-specifically modified. That is, in the present invention, a step of reacting a peptide with an antibody to obtain a peptide-modified antibody (antibody modification step) may be provided before the labeling step.
• the antibody used in the present invention may be a polypeptide containing an Fc region, and may be a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferable.
• Monoclonal antibodies also include artificially modified genetically modified antibodies such as antibody variants (chimeric antibodies, humanized antibodies, etc.).
• the peptide used in the present invention may be a chain peptide or a cyclic peptide as long as it modifies the Fc region of the antibody in a site-specific manner, but a cyclic peptide is preferable. Further, it is preferable to include an amino acid sequence consisting of 13 to 17 amino acid residues represented by the following formula (i). The following description of the amino acid sequence will be described assuming that the left side of the paper surface of the amino acid sequence indicates the N-terminal side and the right side of the paper surface of the amino acid sequence indicates the C-terminal side.
• the binding position of the second atomic group linked to the peptide is not particularly limited as long as it can be click-reacted with the first atomic group, but from the viewpoint of improving reactivity, the N-terminal or C of the peptide is preferable. It has a second atomic group at the terminal, and more preferably has a second atomic group at the N-terminal of the peptide.
• Xa, Xb, Xc and Xd represent a continuous X, a continuous b X, a continuous c X, and a continuous d X, respectively.
• X is an amino acid residue that has neither a thiol group nor a haloacetyl group in the side chain.
• a, b, c and d are each independently an integer of 1 or more and 5 or less, and satisfy a + b + c + d ⁇ 14.
• Xaa1 and Xaa3 are independent of each other.
• Xaa2 is a lysine residue, an arginine residue, a cysteine residue, an aspartic acid residue, a glutamate residue, 2-aminosveric acid, or a diaminopropionic acid, and is preferably modified with a cross-linking agent.
• amino acid residues that can be contained in X in the above formula (i) are derived from amino acids such as glycine, alanine, phenylalanine, proline, aspartic acid, aspartic acid, glutamic acid, arginine, histidine, serine, threonine, tyrosine, and methionine.
• X may be an amino acid residue composed of the same type of amino acid, or may be an amino acid residue composed of different types of amino acids.
• a + b + c + d ⁇ 14 is a condition. Is preferably an integer of 1 or more and 3 or less, b is preferably an integer of 1 or more and 3 or less, c is preferably an integer of 3 or more and 5 or less, and d is preferably an integer of 1 or more and 3 or less.
• Xaa1 and Xaa3 are amino acid residues derived from amino acids having a thiol group in the side chain, and the amino acids may be the same or different.
• amino acids having a thiol group in the side chain include cysteine and homocysteine. It is preferable that such an amino acid residue is bound by a disulfide bond or a sulfide group is bound via a structure represented by the following formula (4). In the formula (4), the broken line portion indicates the bonding portion with the sulfide group.
• Xaa1 and Xaa3 are amino acid residues derived from an amino acid having a thiol group in the side chain and an amino acid derived from an amino acid having a haloacetyl group in the side chain, instead of the combination described above. It may be a residue. These are bound via a thioether bond. The terminal of the haloacetyl group is substituted with a halogen such as iodine, and the halogen is eliminated by the reaction with the thiol group in the other side chain to form a thioether bond.
• a halogen such as iodine
• Specific amino acid sequences of the peptides represented by the formula (i) include, for example, the peptides described in International Publication No. 2016/186206, International Publication No. 2017/217347, and International Publication No. 2018/230257. Can also be used.
• amino acid sequence of the peptide it is preferable to have any one of the following sequences (1) to (14) as the amino acid sequence of the peptide, and the following sequences (1), (2), (13) or ( It is more preferable to have 14).
• the binding affinity between the peptide and the antibody for example, human IgG
• (Xaa2) indicates a lysine residue, a cysteine residue, an aspartic acid residue, a glutamate residue, 2-aminosveric acid, or diaminopropionic acid, preferably. It has been modified with a cross-linking agent, and both (Xaa1) and (Xaa3) show homocysteine residues.
• amino acids other than (Xaa1), (Xaa2) and (Xaa3) are represented by one-letter abbreviations.
• the peptide used in the present invention uses a combination of natural amino acids and amino acids regardless of unnatural amino acids, and is a peptide such as a liquid phase synthesis method, a solid phase synthesis method, an automatic peptide synthesis method, a gene recombination method, or a phage display method. It can be manufactured by subjecting it to a synthetic method. In synthesizing the peptide, the functional groups of the amino acids used may be protected, if necessary. These can be performed, for example, according to the methods described in International Publication No. 2017/217347 Pamphlet and International Publication No. 2018/230257 Pamphlet.
• a peptide having a desired amino acid sequence is obtained by the above method, and then the peptide is used as a solubilizing agent and a reducing agent, and if necessary, an acid.
• examples thereof include a method in which the solution is dissolved in the added solution, an organic solvent solution of the atomic group containing an azido group or TCO is added to the solution as the second atomic group, and the solution is stirred at room temperature to introduce the solution.
• an azide group-containing atomic group As the second atomic group, a commercially available azide group-introducing reagent is used, and the azide group is directly introduced into the N-terminal or C-terminal of the peptide according to a conventional method, or the above-mentioned An atomic group containing an azide group can be introduced through the linker structure.
• the azide group-introducing reagent used include silyl azide, phosphoric acid azide, alkylammonium azide, inorganic azide, sulfonyl azide, and PEG azide.
• an atomic group containing TCO as the second atomic group, whether TCO is directly introduced into the N-terminal or C-terminal of the peptide according to a conventional method using a commercially available click chemistry reagent containing TCO. , Or an atomic group containing TCO can be introduced via the linker structure described above.
• the method of binding a peptide and an antibody to obtain a peptide-modified antibody can be carried out using, for example, a cross-linking agent.
• the cross-linking agent is a chemical substance for covalently linking a peptide and an antibody, and examples thereof include a succinimidyl group such as disulfide imidazole glutarate (DSG) and disulfide imidazole svelate (DSS).
• a cross-linking agent preferably containing 2 or more, a cross-linking agent consisting of a compound containing 2 or more imidic acid moieties such as dimethyl adipimide acid or a salt thereof, and dimethyl 3,3'-dithiobispropionimide acid, dithiobiskucinimi.
• Examples thereof include compounds having a disulfide bond such as zylpropionic acid or those composed of salts thereof.
• a cross-linking reaction can occur between the amino acid residue of Xaa2 in the peptide and the antibody.
• the site where the cross-linking reaction occurs in the antibody is a site between the amino acid residue of Xaa2 and at least one of Lys246 residue and Lys248 residue according to Eu numbering in trastuzumab when human IgG (for example, trastuzumab) is used as the antibody. It specifically binds via a crosslinked structure.
• Lys residues are present in the Fc region of human IgG, and even for antibodies other than trastuzumab, those skilled in the art can align the amino acid sequences of the antibodies and identify the corresponding Lys residues.
• the method for binding the peptide to the antibody is carried out, for example, by dispersing the above-mentioned peptide, antibody, cross-linking agent, and catalyst, if necessary, in an appropriate buffer solution in an environment of 10 ° C. or higher and 30 ° C. or lower. be able to.
• the reaction time can be 10 minutes or more and about 2 hours.
• the molar ratio of the peptide to the antibody during the reaction can be in the range of 1: 1 to 20: 1 for the peptide: antibody.
• the peptide-modified antibody obtained through the above steps may have at least one peptide molecule bound to one antibody (preferably an antibody having an immunoglobulin class of IgG), but the activity of the antibody itself (antigen). From the viewpoint of maintaining cognitive action, neutralizing action, complement activation action, opsonin action), it is preferable that the peptide is site-specifically bound to the Fc region (constant region) of the antibody. It is more preferable that the modified antibody has one or two peptides bound to one antibody molecule.
• the peptide-modified antibody includes an antibody in which one peptide molecule is bound to one antibody molecule (hereinafter referred to as “monovalent antibody”) and an antibody in which two peptide molecules are bound to one antibody molecule (hereinafter referred to as “divalent antibody”). Although it is a mixture containing "antibody”) in an arbitrary ratio, it may be used as it is in the subsequent steps, such as a filtration filter, a membrane filter, a column filled with various fillers, various chromatographys, etc. After separating and purifying the unmodified antibody, the monovalent antibody, and the divalent antibody by the method, only the antibody having any valence may be subjected to the subsequent steps.
• the mixture may be subjected to the subsequent steps as a mixture containing these.
• any of the above purification methods may be used for separation and purification, but it is preferable to use a column packed with various fillers. It is more preferable to use a column packed with a packing material suitable for separation and purification of proteins such as antibodies.
• a metal nuclide that emits ⁇ -rays, ⁇ -rays, ⁇ -rays, or a combination thereof can be used.
• nuclei of such radioactive metals include alkali metals, alkaline earth metals, lanthanoids, actinides, transition metals, and radioisotopes of metals other than these metals.
• 44 Sc, 51 Cr, 57 Co, 58 Co, 60 Co, 59 Fe, 67 Ga, 68 Ga as nuclides of radiometals, from the viewpoint of being commercially available and improving complex formation.
• radioactive metals can be produced according to a conventional method, and it is preferable to obtain them as a solution containing the radioactive metals in an ionized manner.
• alpha-emitting radionuclide as a radioactive metal or beta - it is preferable to use a line-emitting nuclides.
• the ⁇ -ray emitting nuclide may be any nuclide that emits ⁇ -rays in the process of decay of the radioactive metal.
• 212 Bi, 213 Bi, 227 Th or 225 Ac or the like is preferably used, and more preferably 227 Th or 227 Th or 225 Ac. It is 225 Ac, more preferably 225 Ac.
• the ⁇ - ray emitting nuclide may be a nuclide that emits ⁇ - rays in the process of decay of a radioactive metal .
• a radioactive metal e.g. 60 Co, 59 Fe, 64 Cu, 67 Cu, 89 Sr, 90 Y, 99m Tc, 103 Ru, 153 Sm, 165 Dy, 166 Ho, 177 Lu, 186 Re, 188 Re, 198 Au, 203 Hg, 212 Bi, 213 Bi or 212 Pb, etc.
• 64 Cu, 67 Cu, etc. 89 Sr, 90 Y or 177 Lu is used.
• a radioactive metal-labeled antibody When a radioactive metal-labeled antibody is used for the purpose of diagnosing a disease or detecting a lesion, a ⁇ + ray-emitting nuclide, an electron-capturing decay nuclide, or a ⁇ -ray emitting nuclide is used as the radioactive metal from the viewpoint of improving diagnostic performance. Is preferable.
• the ⁇ + ray emitting nuclide may be any nuclide that emits positrons in the process of decay of the radioactive metal, and 44 Sc, 58 Co, 68 Ga, 64 Cu or 89 Zr or the like is preferably used, and more preferably 64 Cu or 89. Zr.
• the electron-capture decay nuclide may be a nuclide that emits Auger electrons or characteristic X-rays in the process of decay of a radioactive metal, and may be 51 Cr, 57 Co, 58 Co, 67 Ga, 68 Ga, 64 Cu, 89 Zr, 111 In. , 186 Re, 201 Tl or 197 Hg, etc. are preferably used.
• the ⁇ -ray emitting nuclide may be any nuclide that emits ⁇ -rays by ⁇ -decay, and 99 m Tc, 68 Ga or 201 Tl is preferably used as the nuclide that emits ⁇ -rays by ⁇ -decay.
• a radioactive metal coordinated to a radioactive metal complex in the ionic state is selected based on the ionic radius
• 67 Ga, 68 Ga, 64 Cu, 67 are selected as the radioactive metal having an ionic radius of about 70 to 130 pm.
• the above formula (A) or (D) to (I) is used. It is preferable to use a ligand having a chelating portion having the structure shown, and more preferably a ligand having a chelating portion having the structure represented by any of the above formulas (A), (D) or (F) is used. , It is more preferable to use a ligand having a chelating portion having a structure represented by the above formula (A).
• the above formula (A), (C) or (K) is used as the ligand. It is preferable to use a ligand having a chelating part having a structure represented by any of the above, and it is more preferable to use a ligand having a chelating part having a structure represented by the above formula (A).
• any class of IgG, IgA, IgM, IgD or IgE can be used without particular limitation, and among these, IgG is the main class in the secondary response of the immune response. It is a class, is most abundant in blood, and is preferably used because of its high recognition specificity for antigens.
• mammalian IgG is preferable, and specific mammals include primates such as humans and chimpanzees; experimental animals such as rats, mice and rabbits; domestic animals such as pigs, cows, horses, sheep and goats; and dogs and dogs. Examples include pet animals such as cats.
• human IgG or rabbit IgG is further preferable, and human IgG is even more preferable.
• the subclass of these antibodies is also not particularly limited.
• human IgG when used, it can be at least one of IgG1, IgG2, IgG3 and IgG4, and is preferably IgG1, IgG3 or IgG4.
• the radioactive metal-labeled antibody obtained through the above-mentioned production method is one in which a specific site of the antibody is specifically modified with a peptide.
• the radioactive metal complex is directly or indirectly linked to the peptide and has a binding site formed between the peptide and the radioactive metal complex, preferably by a click reaction.
• the binding site preferably has a chemical structure derived from the first atomic group contained in the radioactive metal complex and the second atomic group linked to the peptide.
• the binding site may have a structure containing a substituted triazole skeleton or a structure containing a substituted pyridazine skeleton.
• the structure containing the substituted skeleton for example, a structure containing at least one of a substituent, an aliphatic ring and an aromatic ring is bonded to a triazole skeleton or a pyridazine skeleton, and a bond with a modified portion or a chelating portion is used.
• a structure containing at least one of a substituent, an aliphatic ring and an aromatic ring is bonded to a triazole skeleton or a pyridazine skeleton, and a bond with a modified portion or a chelating portion is used.
• Examples thereof include those having a site and a binding site for a peptide.
• the specific structure of the bond site is as follows, for example, when the first atomic group and the second atomic group are a combination of an atomic group containing DBCO and an atomic group containing an azide group, the reaction reagent used is used.
• a structure containing the triazole skeleton represented by the formula (10a) or the formula (10b) is formed, and these may be contained in an arbitrary ratio because they are isomers.
• the first atomic group and the second atomic group are a combination of an atomic group containing 1,2,4,5-tetrazine and an atomic group containing TCO, the following formula (depending on the reaction reagent used)
• a structure containing the pyridazine skeleton shown in 10c) is formed.
• R 1A indicates a binding site with a modified part or a chelating part
• R 2A indicates a binding site with a peptide
• one of R 3A and R 4A shows a hydrogen atom, a methyl group, a phenyl group or a pyridyl group, the other shows a binding site with a modified part or a chelating part
• R 5A shows a binding site with a peptide. Indicates the site.
• a radiopharmaceutical composition containing the radiometal-labeled antibody as an active ingredient can be prepared as it is or after purification thereof.
• the radiopharmaceutical composition refers to a composition containing a radiometal-labeled antibody or a derivative thereof and in a form suitable for administration into a living body.
• the radiopharmaceutical composition can be produced, for example, by dissolving a radiometal-labeled antibody produced by the above method in a solvent containing water as a main component and substantially isotonic with a living body.
• the radiopharmaceutical composition is preferably in the form of an aqueous solution and may optionally contain other pharmaceutically acceptable ingredients.
• the radiopharmaceutical composition is orally or parenterally administered to a living body such as intravenously, subcutaneously, intraperitoneally or intramuscularly, and is used for treating a disease, diagnosing a disease, detecting a lesion, or the like.
• Examples of the substituents that can be substituted with the above formulas (A) to (J) and the triazole skeleton-containing structure and the pyridazine skeleton-containing structure include a halogen atom, a saturated or unsaturated alkyl group, a hydroxy group, an aldehyde group, a carboxy group, and an acyl group. Examples thereof include a group, an amino group, a nitro group, an ester group, an isothiocyanate group, a thioxy group, a cyano group, an amide group, an imide group, a phosphoric acid group, a phenyl group, a benzyl group and a pyridyl group. One of these substituents may be substituted alone, or two or more of these substituents may be substituted in combination.
• Examples 1 to 4 (1-1. Complex formation step)
• the structures of the ligands used in this example are shown in the following formulas (L1-1) to (L1-3).
• the DO3A-DBCO represented by the formula (L1-1) is described in Liang Y, Jiang X, Yuan R, Zhou Y, Ji C, Yang L et al. Metabolism-Based Click-Mediated Platform for Special Imaging and Quantification of Cell Surface Sialic Acids. Anal Chem. Jan 3; 89 (1): 538-543. It was synthesized according to the method described in (2017).
• the DOTA-DBCO represented by the formula (L1-2) is Wang H, Wang R, Cai K, He H, Liu Y, Yen J et al.
• the radiochemical purity of the obtained 225 Ac complex was measured by the following method. That is, a part of the 225 Ac complex solution was developed by thin layer chromatography (manufactured by Agent, model number: SGI0001, developing solvent: acetonitrile / water mixed solution (volume ratio 1: 1)), and then a radio ⁇ -TLC analyzer was used. (Made by acetonitrile, MODEL GITA Star). The percentage of the radioactivity (count) of the peak detected near the origin with respect to the detected total radioactivity (count) was defined as the radiochemical purity (%) of the 225 Ac complex. As a result, the radiochemical purity of the 225 Ac complex was 89 to 99%. The obtained 225 Ac complex solution was used as it was in the labeling step.
• a peptide was produced by the method described in International Publication No. 2017/217347 pamphlet to obtain a peptide containing 17 amino acid residues represented by the following formula (P3).
• the amino acid sequence of this peptide, Xaa2 of SEQ ID NO: (2) have the same sequence as a lysine residue, the side chain terminal amino group of a lysine residue has been modified by the structure represented by R 1.
• cysteine residues are disulfide-bonded to each other, and the N-terminal of the peptide is ethyl azide as an atomic group containing an azide group, which is a second atomic group, via a linker structure having diglycolic acid and eight PEGs.
• a linker structure having diglycolic acid and eight PEGs. was combined.
• a 0.05 mol / L sodium acetate buffer (pH 3.5) containing 0.15 mol / L sodium chloride was flown to recover the solution containing the monovalent antibody, and the unmodified antibody and monovalent antibody contained in the collected fraction were collected.
• the concentration was adjusted so that the antibody concentration was 17-40 mg / mL.
• the molar ratio between and the second atomic group (azido) was about 1: 1, respectively.
• the reaction rate (%) of the 225 Ac complex-labeled antibody of the example when unpurified is shown in Table 1 below.
• the reaction rate (%) means the radiochemical purity (%) of the 225 Ac complex-labeled antibody with respect to the labeling rate (%) in the complex formation step, and the labeling rate (%) is based on the charged radioactivity amount. It means the amount of radioactivity (%) of the 225 Ac complex.
• a solution of the 225 Ac complex-labeled antibody obtained by reacting at 37 ° C. for 2 hours was purified using an ultrafiltration filter (manufactured by Merck, model number: UFC505096).
• the radiochemical purity (RCP) and radiochemical yield (RCY) of the purified 225 Ac complex-labeled antibody are shown in Table 1 below.
• the method for measuring the radiochemical purity and radiochemical yield of the 225 Ac complex-labeled antibody was as follows. That is, thin layer chromatography (manufactured by Agent, model number: SGI0001, developing solvent is a mixed solution of acetonitrile: 0.1 mmol / L EDTA solution (volume ratio 1: 1)) is applied to a radio ⁇ -TLC analyzer (manufactured by raytest, MODEL GITA Star). ), The percentage of the radioactivity (count) of the peak detected near the origin with respect to the detected total radioactivity (count) was defined as the radiochemical purity (%).
• the total radioactivity added at the start of the labeling process ( ⁇ -ray spectrometer (Ge semiconductor detector: GMX10P4-70 (manufactured by ORTEC), multi-channel analyzer: M7-000 (manufactured by Seiko Easy and G), data processing).
• the percentage of the radioactivity recovered after purification (the amount of radioactivity calculated from the count measured by the ⁇ -ray spectrometer in the same manner as described above) was defined as the radiochemical yield (%).
• Example 5 (1-1. Complex formation step)
• a 225 Ac complex solution was obtained in the same manner as in Example 1 except that a ligand having a structure shown in the following formula (L2) was used.
• DOTA-PEG7-Tz represented by the formula (L2) is described in Poty S, Membrenno R, Glasser JM, Ragupathi A, Scholz WW, Zeglis BM et al. The influence election-demand Diels-Alder reaction as a new methodology for the synthesis of 225Ac-labelled radioimmunoconjutes. Chem Commun (Camb). Mar 8; 54 (21): 2599-2602. (2016).
• the peptide used in this example was a peptide consisting of 17 amino acid residues represented by the following formula (P4).
• the amino acid sequence of this peptide was the same as the sequence in which Xaa2 of SEQ ID NO: (2) was a lysine residue, and the side chain terminal amino group of the lysine residue was modified with the structure shown by R2.
• the thiol groups of the two cysteine residues are linked by a linker represented by the above formula (4), and the N-terminal of the peptide is five PEGs and thiol groups in the side chain when viewed from the N-terminal side.
• a linker structure having a cysteine residue substituted with the structure represented by R 1 two glutamate residues, and an acetyl group in this order, containing TCO, which is the second atomic group.
• Gly is glycine, Pro is proline, Asp is aspartic acid, Cys is cysteine, Ala is alanine, Tyr is tyrosine, His is histidine, Glu is glutamate, and Leu is. Leucine, Val stands for valine, Trp stands for tryptophan, Ph stands for phenylalanine)
• a human IgG antibody (trastuzumab; manufactured by Roche) mixed with sodium acetate buffer (pH 6) was reacted at room temperature for 30 minutes to obtain a solution containing the peptide-modified antibody.
• the labeling reaction proceeded under mild reaction conditions without excessive heating of the antibody. Moreover, it can be seen that the labeling efficiency of the radioactive metal with respect to the antibody is excellent.
• ligands were dispersed in 0.1 mol / L sodium acetate buffer (pH 6.0) as a solvent to prepare a dispersion containing 1.7 mmol / L of the ligand. 0.005 mL of this dispersion and a 225 Ac ion-containing solution as a radioactive metal source (0.2 mol / L hydrochloric acid aqueous solution, radioactivity concentration 320 to 340 MBq / mL, prepared from Oak Ridge National Laboratory, liquid volume 0.005 mL) 1.
• a reaction solution mixed with 6 to 1.7 MBq (calculated value calculated by attenuation from the amount of radioactivity on the test date and time) was reacted under heating conditions to obtain a 225 Ac complex solution.
• the radiochemical purity of the obtained 225 Ac complex was measured in the same manner as in Example 1 and found to be 90%.
• the obtained 225 Ac complex solution was used as it was in the labeling step.
• the reaction rate (%) of the 225 Ac complex-labeled antibody when unpurified is shown in Table 2 below.
• the reaction rate (%) means the radiochemical purity (%) of the 225 Ac complex-labeled antibody with respect to the labeling rate (%) in the complex formation step, and the labeling rate (%) is based on the charged radioactivity amount. It means the amount of radioactivity (%) of the 225 Ac complex.
• a solution of the 225 Ac complex-labeled antibody obtained by reacting at 37 ° C. for 2 hours was purified using an ultrafiltration filter (manufactured by Merck, model number: UFC505096).
• the radiochemical purity (RCP) and radiochemical yield (RCY) of the purified 225 Ac complex-labeled antibody are shown in Table 2 below.
• Example 8 Production of Tmab using 89 Zr-labeled DOTAG A-DBCO (1. Complex forming step)
• the structure of the ligand used in this example is the same as the above formula (L1-4).
• This ligand was dispersed in DMSO as a solvent to prepare a dispersion containing 0.33 mmol / L of the ligand. 0.030 mL of this dispersion and 60 MBq of 89 Zr ion-containing solution (0.1 mol / L hydrochloric acid aqueous solution, radioactivity concentration 181 MBq / mL, prepared from Nippon Mediphysics Co., Ltd., liquid volume 0.33 mL) as a radioactive metal source.
• the radiochemical purity of the obtained 89 Zr complex was measured by the following method. That is, a part of the 89 Zr complex solution was developed by thin layer chromatography (manufactured by Agent, model number: SGI0001, developing solvent: acetonitrile / water mixed solution (volume ratio 1: 1)), and then a radio ⁇ -TLC analyzer was used. (Made by acetonitrile, MODEL GITA Star PS). The percentage of the radioactivity (count) of the peak detected near the origin with respect to the detected total radioactivity (count) was defined as the radiochemical purity (%) of the 89 Zr complex. As a result, the radiochemical purity of the 89 Zr complex was 90%. The obtained 89 Zr complex solution was used as it was in the labeling step.
• the reaction rate (%) of the 89 Zr complex-labeled antibody of the example when unpurified is shown in Table 3 below.
• the reaction rate (%) means the radiochemical purity (%) of the 89 Zr complex-labeled antibody with respect to the labeling rate (%) in the complex formation step, and the labeling rate (%) is based on the charged radioactivity amount. It means the amount of radioactivity (%) of the 89 Zr complex.
• a solution of 89 Zr complex-labeled antibody obtained by reacting at 37 ° C. for 2 hours was purified using an ultrafiltration filter (manufactured by Merck, model number: UFC550906).
• the radiochemical purity (RCP) and radiochemical yield (RCY) of the purified 89 Zr complex-labeled antibody are shown in Table 3 below.
• Example 9 Production of Tmab using 89 Zr-labeled DOTA-DBCO (1-1. Complex formation step)
• the structure of the ligand used in this example is the same as the above formula (L1-2).
• This ligand was dispersed in DMSO as a solvent to prepare a dispersion containing 1 mmol / L of the ligand.
• 0.1 mL of this dispersion was mixed with 45 MBq of an 89 Zr ion-containing solution (0.1 mol / L hydrochloric acid aqueous solution, radioactivity concentration 450 MBq / mL, prepared by Okayama University, liquid volume 0.1 mL) as a radioactive metal source.
• an 89 Zr ion-containing solution 0.1 mol / L hydrochloric acid aqueous solution, radioactivity concentration 450 MBq / mL, prepared by Okayama University, liquid volume 0.1 mL
• the reaction solution was reacted under heating conditions to obtain an 89 Zr complex solution.
• the radiochemical purity of the obtained 89 Zr complex was carried out according to Example 1. As a result, the radiochemical purity of the 89 Zr complex was 99%.
• the obtained 89 Zr complex solution was fractionated by high performance liquid chromatography (HPLC) to remove unreacted DOTA-DBCO.
• HPLC high performance liquid chromatography
• the radiochemical yield (HPLC recovery rate) in the step of removing the unreacted substance of the 89 Zr complex-labeled antibody is shown in Table 4 below.
• the method for measuring the radiochemical yield (HPLC recovery rate) is to set the percentage of the amount of radioactivity in the preparative solution to the amount of radioactivity charged at the start of this step as the HPLC recovery rate (%) in the unreacted material removal step. And said.
• a solution of 89 Zr complex-labeled antibody obtained by reacting at 37 ° C. for 2 hours was purified using an ultrafiltration filter (manufactured by Merck, model number: UFC550906).
• the radiochemical purity (RCP) and radiochemical yield (RCY) of the purified 89 Zr complex-labeled antibody are shown in Table 4 below.

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