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
  • A61K51/08—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
  • A61K51/1027—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 against receptors, cell-surface antigens or cell-surface determinants
  • A61K51/103—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 against receptors, cell-surface antigens or cell-surface determinants against receptors for growth factors or receptors for growth regulators
• • 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
  • A61K51/1045—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 against animal or human tumor cells or tumor cell determinants
• • 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
  • A61K51/1045—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 against animal or human tumor cells or tumor cell determinants
  • A61K51/1051—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 against animal or human tumor cells or tumor cell determinants the tumor cell being from breast, e.g. the antibody being herceptin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
  • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype

Definitions

• the present invention relates to a method for producing a radiolabeled antibody, a radiolabeled antibody, and a radiopharmaceutical.
• ADCs antibody-drug conjugates
• Patent Documents 1 to 3 disclose the CCAP (Chemical Conjugation by Affinity Peptide) method, which is a site-specific modification method for preparing conjugates of antibodies and drugs, etc.
• an antibody modification reagent containing an IgG-binding peptide that specifically binds to the Fc region of IgG is reacted with the antibody.
• IgG-BP IgG-binding peptide
• Certain amino acid residues of the IgG-BP are modified with N,N'-disuccinimidyl glutarate (DSG) or dithiobis(succinimidyl propionate) (DSP) and contain an N-hydroxysuccinimide (NHS) group.
• DSG N,N'-disuccinimidyl glutarate
• DSP dithiobis(succinimidyl propionate)
• NHS N-hydroxysuccinimide
• the structure of the Fc region is symmetric, so there are usually two IgG-BP binding sites in the Fc region. Therefore, the CCAP method can produce bivalent modified antibodies in which IgG-BP is bound to both IgG-BP binding sites, and monovalent modified antibodies in which IgG-BP is bound to one IgG-BP binding site.
• Patent Document 2 discloses that an antibody was labeled with a radioactive metal nuclide using the CCAP method to obtain a monovalent radiolabeled antibody modified with one molecule of chelate-binding peptide per antibody molecule, and a bivalent radiolabeled antibody modified with two molecules of chelate-binding peptide per antibody molecule.
• Patent Document 2 further discloses that PET (Positron Emission Tomography) imaging of a cancer-bearing mouse was performed using each of monovalent [ 89 Zr] trastuzumab and bivalent [ 89 Zr] trastuzumab.
• the present invention has been made in consideration of the above circumstances, and aims to provide a method for producing a radiolabeled antibody that has a higher blood retention than bivalent radiolabeled antibodies produced using the conventional CCAP method, as well as a radiolabeled antibody and a radiopharmaceutical.
• the method for producing a radiolabeled antibody according to the first aspect of the present invention comprises the steps of: The following formula (1): P1-SY-B Formula (1)
• P1 is a labeling moiety that is or can be labeled with a radioisotope
• S is a spacer
• Y is a reactive site that reacts with IgG
• B is an antibody binding site that includes an IgG-binding peptide that specifically binds to the Fc region of IgG. or a salt thereof with IgG
• Y in the formula (1) has a group represented by the following formula I.
• R 1 is a substituent that gives R 1 -H a pKa of 4 to 14
• R2 is bonded to S in the formula (1)
• R3 is bonded to B in the formula (1)
• R4 is H or a substituted or unsubstituted alkyl group having 1 to 8 carbon atoms.
• the radiolabeled antibody according to the second aspect of the present invention comprises: The following formula (4): (P2-S)n-Ab Formula (4) (In formula (4), P2 comprises a radioisotope; S is a spacer, Ab is IgG, n is 1 or 2. As shown in S in the formula (4) contains Lys-Gly-Gly, and the amino group of the side chain of Lys in S in the formula (4) is substituted with P2; In the formula (4), S specifically binds to Lys in the Fc region of IgG.
• the radiolabeled antibody according to the third aspect of the present invention comprises: The following formula (4): (P2-S)n-Ab Formula (4) (In formula (4), P2 comprises a radioisotope; S is a spacer, Ab is IgG, n is 1 or 2. As shown in S in the formula (4) is selected from the group consisting of a polyethylene glycol chain, an alkylene chain, and a combination thereof; In the formula (4), S specifically binds to Lys in the Fc region of IgG.
• the radiopharmaceutical according to the fourth aspect of the present invention comprises: The radiolabeled antibody according to the second or third aspect of the present invention is contained as an active ingredient.
• radiolabeled antibodies and radiopharmaceuticals of the present invention have a higher retention in the blood than bivalent radiolabeled antibodies produced using the conventional CCAP method.
• FIG. 1 is a schematic diagram showing a reaction for modifying an antibody with a compound according to an embodiment of the present invention.
• FIG. 2 shows an elution chromatogram obtained by liquid chromatography mass spectrometry (LC-MS) of the antibody solution according to Example 1.
• FIG. 2 is a diagram showing the results of mass spectrometry of an IgG peak in an antibody solution before the addition of compound A according to Example 1.
• FIG. 1 shows the results of mass spectrometry of the IgG peak in the reaction solution after 2 hours of reaction in Example 1.
• FIG. 1 shows corrected count values regarding the antigen binding activity of Example 1 and Comparative Example 1 in Test Example 1.
• FIG. 13 is a graph showing the time course of the administered radioactivity accumulation rate per unit weight of blood (% ID/g) in mice according to Test Example 2.
• FIG. 13 is a graph showing the time course of the accumulation rate of administered radioactivity per unit weight in the liver of a mouse in Test Example 2.
• FIG. 13 is a graph showing the time course of the accumulation rate of administered radioactivity per unit weight in the kidney of mice in Test Example 2.
• FIG. 13 is a graph showing the time course of the accumulation rate of administered radioactivity per unit weight in the femur of a mouse in Test Example 2.
• FIG. 13 is a graph showing the time course of the accumulation rate of administered radioactivity per unit weight in the thigh muscle of a mouse in Test Example 2.
• FIG. 13 is a graph showing the time course of the accumulation rate of administered radioactivity per unit weight of the spleen of a mouse in Test Example 2.
• FIG. 13 is a diagram showing the results of mass spectrometry of an IgG peak in an antibody solution before the addition of compound A according to Example 2.
• FIG. 1 shows the results of mass spectrometry of an IgG peak in a reaction solution containing compound A according to Example 2.
• FIG. 13 is a diagram showing the results of mass spectrometry of an IgG peak in an antibody solution before the addition of compound B according to Example 2.
• FIG. 1 shows the results of mass spectrometry of an IgG peak in a reaction solution containing compound B according to Example 2.
• FIG. 13 is a diagram showing the results of mass spectrometry of an IgG peak in an antibody solution before addition of compound C according to Example 2.
• FIG. 1 shows the results of mass spectrometry of an IgG peak in a reaction solution containing compound C according to Example 2.
• FIG. 13 is a diagram showing the results of mass spectrometry of an IgG peak in an antibody solution before addition of compound D according to Example 2.
• FIG. 1 shows the results of mass spectrometry of an IgG peak in a reaction solution containing compound D according to Example 2.
• FIG. 13 is a diagram showing the results of mass spectrometry of an IgG peak in an antibody solution before the addition of compound A in Example 3.
• FIG. 1 shows the results of mass spectrometry of an IgG peak in a reaction solution containing compound A according to Example 3.
• FIG. 13 is a diagram showing the results of mass spectrometry of an IgG peak in an antibody solution before the addition of compound B according to Example 3.
• FIG. 13 is a diagram showing the results of mass spectrometry of an IgG peak in a reaction solution containing compound B according to Example 3.
• FIG. 13 is a diagram showing the results of mass spectrometry of an IgG peak in an antibody solution before the addition of compound C according to Example 3.
• FIG. 1 shows the results of mass spectrometry of an IgG peak in a reaction solution containing compound A according to Example 3.
• FIG. 13 is a diagram showing the results of mass spectrometry of an IgG peak in an antibody solution before the addition of compound B according to Example 3.
• FIG. 13 is a diagram showing the results of mass spectrometry of an IgG peak in a reaction solution containing compound C according to Example 3.
• FIG. 1 shows the results of size exclusion chromatography (SEC) analysis in Test Example 4.
• FIG. 13 is a diagram showing corrected count values regarding the antigen-binding activity of the radioconjugate of Example 2 in Test Example 5.
• FIG. 13 is a graph showing the time course of accumulation of the radioactive conjugate of Example 2 in a tumor in Test Example 6.
• FIG. 13 is a graph showing the time course of accumulation of the radioactive complex of Example 2 in the heart in Test Example 6.
• FIG. 13 is a graph showing the time course of accumulation of the radioactive complex of Example 2 in the liver in Test Example 6.
• FIG. 1 shows the results of size exclusion chromatography (SEC) analysis in Test Example 4.
• FIG. 13 is a diagram showing corrected count values regarding the antigen-binding activity of the radioconjugate of Example 2 in Test Example 5.
• FIG. 13 is a graph showing the
• FIG. 13 is a graph showing the time course of accumulation of the radioactive conjugate of Example 2 in the kidney in Test Example 6.
• FIG. 13 is a diagram showing corrected count values regarding the antigen-binding activity of the radioconjugate of Example 3 in Test Example 7.
• FIG. 13 is a graph showing the time course of accumulation of the radioactive conjugate of Example 3 in a tumor in Test Example 8.
• FIG. 13 is a graph showing the time course of accumulation of the radioactive complex of Example 3 in the heart in Test Example 8.
• FIG. 13 is a graph showing the time course of accumulation of the radioactive complex of Example 3 in the liver in Test Example 8.
• FIG. 13 is a graph showing the time course of accumulation of the radioconjugate of Example 3 in the kidney in Test Example 8.
• FIG. 13 is a diagram showing corrected count values regarding the antigen-binding activity of the radioconjugate of Example 3 in Test Example 7.
• FIG. 13 is a graph showing the time course of accumulation of the radioactive conjugate of Example 3 in a tumor in Test Example 8.
• FIG. 13 is a diagram showing corrected count values regarding the antigen-binding activity of the radioconjugate of Example 4 in Test Example 10.
• FIG. 13 is a graph showing the change over time in tumor volume in tumor-bearing mice administered the radioactive complex of Example 4 in Test Example 11.
• the method for producing a radiolabeled antibody includes a conjugation step of reacting a compound represented by the following formula (1) or a salt thereof with IgG. P1-SY-B Formula (1)
• P1 is a labeling site that is labeled with a radioisotope or a labeling site that can be labeled with a radioisotope.
• the radioisotope is not particularly limited as long as it is an element that emits radiation and can be traced as a radiolabeled antibody.
• a radioactive nuclide that emits ⁇ -rays, ⁇ -rays, ⁇ -rays, or a combination thereof can be used.
• radioisotopes include radioisotopes of halogen elements, alkali metals, alkaline earth metals, lanthanides, actinides, transition metals, and other metals.
• radioisotope a radioactive metal nuclide is preferred. From the viewpoint of commercial availability and improving the complex forming property, 44 Sc, 51 Cr, 57 Co, 58 Co, 60 Co, 59 Fe, 67 Ga, 68 Ga, 64 Cu, 67 Cu, 89 Sr, 89 Zr, 90 Y, 99m Tc, 103 Ru, 111 In, 153 Sm, 165 Dy, 166 Ho, 177 Lu, 186 Re , 188 Re, 198 Au, 201 Tl, 197 Hg, 203 Hg, 212 Bi, 213 Bi, 212 Pb, 227 Th, or 225
• radioisotopes include radioactive fluorine atom ( 18 F), radioactive chlorine atom ( 36 Cl), radioactive bromine atom ( 75 Br, 76 Br, 77 Br, 80 Br), radioactive iodine atom ( 123 I, 124 I, 125 I), and radioactive halogen atom such as radioactive astatine atom ( 221 At), Al
• P1 When the labeling site is labeled with a radioisotope, P1 has a reactive functional group, and after the conjugation step, the labeling site is labeled with the radioisotope.
• reactive functional groups include imidazole groups, hydroxy groups, amino groups, thiol groups, halogen atoms, sulfonate ester groups, epoxy groups, isocyanate groups, azide groups, vinyl groups, maleimide groups, ethynyl groups (-C ⁇ CH), dienes, alkynes, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctyne (DBCO) groups, trans-cyclooctene (TCO), tetrazine, and the like.
• the reactive functional group is an azide group
• the radioisotope is added to P1 by a click reaction with an alkyne or DBCO group, etc.
• S in formula (1) is a spacer.
• the spacer may be selected from the group consisting of a substituted or unsubstituted alkyl group having 1 to 8 carbon atoms, a substituted or unsubstituted alkenyl group having 2 to 8 carbon atoms, a substituted or unsubstituted alkynyl group having 2 to 8 carbon atoms, a substituted or unsubstituted peptide chain having 2 to 50 amino acid residues, a substituted or unsubstituted polymer chain having a degree of polymerization of 2 to 50, and combinations thereof.
• the number of amino acid residues in the peptide chain is preferably 2 to 30, 2 to 20, 2 to 10, or 2 to 5, and more preferably 3.
• the amino acid residues in the peptide chain may be those of natural amino acids or artificial amino acids, and may be, for example, one or more glycine residues or one or more lysine residues, or a combination thereof.
• a specific example of the peptide chain is Lys-Gly-Gly.
• the polymer chain is not particularly limited and may be any known polymer chain, such as PEG, and may specifically be a PEG chain with a degree of polymerization of 2 to 50.
• the substituted peptide chain includes those in which a substituent is bonded to the main chain or side chain.
• the substituted polymer chain includes those in which a substituent is bonded to the main chain or side chain.
• the spacer includes Lys-Gly-Gly, and the amino group in the side chain of Lys is substituted with P1 in the above formula (1).
• Alkyl group means a saturated hydrocarbon group.
• Alkyl groups include cyclic (including fused bicyclic) alkyl groups that satisfy the carbon number requirement, straight-chain and branched-chain alkyl groups, and straight-chain or branched-chain alkyl groups substituted with a cyclic alkyl group.
• alkyl groups include methyl, ethyl, n-propyl, iso-propyl, cyclopropyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-ethylpropyl, n-hexyl, cyclohexyl, cyclooctyl, and 1-methyl-2-ethylpropyl.
• Alkenyl group means a hydrocarbon group having at least one double bond. Alkenyl includes both straight-chain and branched-chain alkenyl groups. Examples of alkenyl include ethenyl, n-propenyl, iso-propenyl, n-butenyl, iso-butenyl, sec-butenyl, t-butenyl, n-pentenyl, 1,1-dimethylpropenyl, 1,2-dimethylpropenyl, 2,2-dimethylpropenyl, 1-ethylpropenyl, 2-ethylpropenyl, n-hexenyl, and 1-methyl-2-ethylpropenyl.
• Alkynyl group means a hydrocarbon group having at least one triple bond.
• Alkynyl includes both straight-chain and branched-chain alkynyl groups. Examples of alkynyl include ethynyl, n-propynyl, iso-propynyl, n-butynyl, iso-butynyl, sec-butynyl, t-butynyl, and n-pentynyl.
• the chain length of the PEG chain contained in the spacer is not particularly limited.
• the degree of polymerization of the PEG chain is, for example, 2 to 30, 3 to 28, 3 to 27, 3 to 25, or 3 to 24.
• the spacer may have an alkylene chain (-C m H2 m -) having m carbon atoms.
• the carbon number m of the alkylene chain is, for example, 1 to 10, 1 to 8, 1 to 6, or 1 to 4.
• the spacer may also contain a PEG chain and an alkylene chain, and for example, the PEG chain bonded to Y may be bonded to P1 via an alkylene chain.
• the degree of polymerization of the PEG chain is n
• the above formula (1) is represented by P1-C m H2 m -(PEG) n -Y-B.
• IgG may be IgG of mammals, for example, primates such as humans and chimpanzees, laboratory animals such as rats, mice and rabbits, livestock animals such as pigs, cows, horses, sheep and goats, and pets such as dogs and cats.
• IgG chimeric antibodies, humanized antibodies or human antibodies of monoclonal antibodies are more preferable.
• the subclasses of these antibodies can be used without any particular restrictions.
• it is human IgG (IgG1, IgG2, IgG3 or IgG4).
• IgG When human IgG is used as IgG, at least one of IgG1, IgG2, IgG3 and IgG4 can be used.
• IgG may be rabbit IgG.
• the antibody may be a full-length antibody or an antibody fragment (F(ab')2, Fab', Fab, Fv, single-chain antibody), preferably a full-length antibody.
• Y in formula (1) has a group represented by the following formula I.
• R 1 is a substituent that gives R 1 -H an acid dissociation constant (pKa) of 4 to 14, 4 to 12, or 4 to 10, preferably 5 to 9.
• pKa is the pKa at room temperature (e.g., 25°C).
• the above compound becomes an activated intermediate by elimination of R 1.
• the pKa of R 1 -H determines the ease of elimination, that is, the ease of transition to an activated intermediate. If the pKa is high, the transition becomes difficult, and if the pKa is low, the transition becomes easy, but if the pKa is too low, the compound is unstable and may be hydrolyzed.
• R 1 -H is a phenol.
• R 1 -H examples include salicylic acid, phenol, 4-fluorophenol, 4-nitrophenol, 2,6-dichlorophenol, N-hydroxysuccinimide, 2,3,5,6-tetrafluorophenol, pentafluorophenol, aminophenol, and dinitrophenol.
• R 1 -H is 4-nitrophenol and R 1 is a nitrophenoxy group.
• R2 in the above formula I is bound to S in the above formula (1).
• R3 in the above formula I is bound to B in the above formula (1).
• B is an antibody binding site containing IgG-BP that specifically binds to the Fc region of IgG.
• the "IgG Fc region” refers to a C-terminal fragment obtained by treating IgG with the protease papain.
• R 3 may be IgG-BP.
• R 3 may also have a linker and IgG-BP, and R 3 may be *-(linker)-(IgG-BP) where * is a carbon of the benzene ring of formula I.
• the IgG-BP in R3 is bound to the carbon of the benzene ring or the linker of formula I through the main chain or the side chain of any amino acid residue.
• the IgG-BP in R3 is bound to the carbon of the carbonyl group or the linker of formula I through the main chain or side chain amino group of the N-terminus.
• B in formula (1) is represented by any one of the following formulas II, III and IV, with IgG-BP being R 3a .
• * represents a carbon atom of the benzene ring in formula I.
• IgG-BP is a peptide that binds to a site selected from Lys246, Lys248, Lys288, Lys290, Lys338, Lys360, Lys414, and Lys439 in Fc according to Eu numbering, or a region adjacent thereto, preferably Lys248 or a region adjacent thereto, or that binds to the binding region of protein A.
• IgG-BP may be a partial peptide of protein A having Fc binding ability, or a variant thereof. Specific examples of such peptides are described in WO 2008/054030, WO 2013/027796, and the above-mentioned Patent Documents 1 to 3, etc.
• IgG-BP can be appropriately prepared according to a known peptide synthesis method, for example, a peptide solid-phase synthesis method or the method described in each of the above-mentioned documents.
• R 3 includes the following (i) to (iii).
• each (Linker) is independently the same or different linker, or absent, wherein the linker is RRRGS (SEQ ID NO: 156), EEGGS (SEQ ID NO: 157), (GSGGS) 1-3 (SEQ ID NOs: 158-160) or (PEG) 1-10 , preferably (PEG) 1-8 or (PEG) 2-10 , more preferably (PEG) 4 ; 1 to 3 X 1 , X 2 , X 3 , X 4 ; 1 to 3
• Dab, Tle, and Cha respectively mean 2,4-diaminobutanoic acid, tert-leucine, and ⁇ -cyclohexyl-L-alanine.
• Ace and t-Bu respectively mean that there is an acetyl group and that there is a tert-butyl group.
• the amino group at the N-terminus may be acetylated.
• a Lys residue may be introduced at an appropriate position near the N-terminus in the Linker.
• IgG-BP contained in the substituent represented by formula (PI) include the following peptides.
• X 1 1-3 is an amino acid sequence represented by (S, G, F, or none)-(D, G, A, S, P, Hcy, or none)-(S, D, T, N, E, or R).
• X 1 1-3 is D, GPD, R, GPR, SPD, GDD, GPS, SDD, RGN, G-Hcy-D, RGP, or GPD.
• X 1 1-3 is D or GPD.
• X2 is A, S, or T.
• X2 is A or T.
• X2 is A.
• X3 is Y or W.
• X3 is Y.
• X4 is H.
• X5 is an amino acid residue selected from A, R, K, C, D, E, L, 2-aminosuberic acid, Dpr, R, F, 2-aminosuberic acid, Dpr, AceOrn, AceDab, Dab, Nle, Nva, Ala(t-Bu), and Cha.
• X5 is K, R, AceOrn.
• X5 is an amino acid residue selected from V, Dab, F, R, L, Nva, Nle, Ala(t-Bu), and Cha.
• X5 is an amino acid residue selected from F, R, L, Nva, Nle, Ala(t-Bu), and Cha.
• X5 is an amino acid residue selected from L, Ala(t-Bu), and Cha.
• X6 is E or N.
• X6 is E.
• X7 is V.
• X 8 1-3 is (S, T, or D)-(H, G, Y, T, N, D, F, Hcy, or none)-(Y, F, H, M, or none).
• X 8 1-3 is T, TFH, S, SFH, THH, TFY, TYH, or T-Hcy-H.
• X 8 1-3 is T or TFH.
• the IgG-BP contained in the substituent represented by formula (PI) may be any one or a combination of two or more of the above conditions, and may be, for example, a peptide that satisfies the conditions described below: [8] and [9], [8] and [17], [9] and [17], [8], [9] and [17], or a combination of these with any one of [10] to [14].
• the amino acid sequence of IgG-BP may be any one of the amino acid sequences shown in SEQ ID NOs: 1 to 151 below.
• X5 is the same as above, and may have an NH2- (Linker)- group at the N-terminus, and may have an -NH2 group or -(Linker) -NH2 group at the C-terminus.
• R3 examples include substituents having the following structures: 60) GSGGS-GPDCAYHRGELVWCTFH-NH 2 (SEQ ID NO: 60) (PEG) 4 -GPDCAYHRGELVWCTFH-NH 2 (SEQ ID NO: 33) 61) GSGGS-DCAYHRGELVWCT- NH2 (SEQ ID NO:61) (PEG) 4 -DCAYHRGELVWCT-NH 2 (SEQ ID NO: 32)
• IgG-BP includes a peptide bound to a functional group Z.
• the substituent represented by formula (PI) may have a functional group Z bound to the tip of (Linker).
• Examples of such a substituent include the following substituents. 62) Acetyl-K(Z)-RRRGS-GPDCAYHKGELVWCTFH-NH 2 (SEQ ID NO: 62) 63) Acetyl-K(Z)-EEGGS-GPDCAYHKGELVWCTFH-NH 2 (SEQ ID NO: 63) 64) Acetyl-K(Z)-(PEG) 4 -GPDCAYHKGELVWCTFH-NH 2 (SEQ ID NO: 64)
• R3 may be a substituent in which a maleimide group, a DBCO group, a tetrazine group, or a TCO group is bonded to the N-terminus or PEG of the following peptides, or a substituent in which an NH2 group is bonded to the C-terminus.
• PII A substituent containing an IgG-BP represented by the following formula (PII), or a substituent containing an IgG-BP consisting of an amino acid sequence in which one or several amino acids have been added, deleted and/or substituted at positions other than X 9 to X 14 in the amino acid sequence of formula (PII): X 9 1-2 NMQX 10 QX 14 RFYEALHDPNLNEEQRNAX 11 IX 12 SIRDDX 13 -(Linker2)
• (Linker2) is (GSGGS) 1-3 (SEQ ID NOs: 158 to 160), (SGSGS) 1-3 (SEQ ID NOs: 161 to 163), or (PEG) 2-10 -Lys (preferably, (PEG) 4 -Lys), SGSGSK (SEQ ID NO: 164), SRRCR (SEQ ID NO: 165), SRRK(Z)R (SEQ ID NO: 166), SRRCRCRRC (SEQ ID NO: 167), SRRK(Z)RRK(Z)RRK(Z) (SEQ ID NO: 168), or (PEG) 1-8 -Lys (preferably, (PEG) 4 -Lys), or is absent;
• X 9 1-2 is selected from the group consisting of GF, AF, VF, LF, IF, MF, PF, FF, WF, KF, Orn-F, CF, DF, EF, ⁇ -alanine-F, 2-ami
• the Cys residue (C) contained in the linker may be bonded to another functional molecule via a maleimide group, if necessary.
• IgG-BP contained in the substituent represented by formula (PII) include the following peptides.
• X 9 is selected from the group consisting of GF, F, K, and Acetyl-K.
• X10 is Q.
• X 11 and X 12 are each independently selected from the group consisting of R, H, and E.
• X11 is R.
• X 12 is R or K(Z) (preferably, Z is azide).
• examples of the substituent represented by formula (PII) include the following substituents (however, a functional group may be bonded to the Lys residue contained therein, if necessary).
• the amino acid sequence of IgG-BP may be any one of the amino acid sequences shown in SEQ ID NOs: 116 to 151, lacking the N-terminal amino acid residues of the amino acid sequences shown in SEQ ID NOs: 72 to 107.
• R3 may be bonded to a carbon of the benzene ring of formula I or to the linker via a Lys residue contained in IgG-BP, particularly via the side chain of X5 described above.
• the side chain of any Lys residue may be bonded to a carbon of the benzene ring of formula I or to a linker.
• any two cysteine residues may form an intramolecular (peptide) bond via a disulfide bond.
• R 4 is H or a substituted or unsubstituted alkyl group having 1 to 8 carbon atoms.
• R 4 is, for example, an alkyl group having 1 to 6, 1 to 4, or 1 to 3 carbon atoms, preferably an ethyl group, and more preferably a methyl group.
• the above compounds can be synthesized by known methods. For example, when synthesizing IgG-BP on a protected peptide resin by solid-phase peptide synthesis, the MeDbz residue is inserted using Fmoc-MeDbz (3-[(9-Fluorenylmethoxycarbonyl)amino]-4-(methylamino)benzoic acid) during peptide elongation, and the skeleton shown in formula (I) above can be constructed at the MeDbz residue site by treating the protected peptide resin with 4-Nitrophenyl Chloroformate.
• Fmoc-MeDbz 3-[(9-Fluorenylmethoxycarbonyl)amino]-4-(methylamino)benzoic acid
• the compound or its salt according to this embodiment does not have an NHS group that is easily hydrolyzed, and therefore is chemically stable and can be stored for a long period of time.
• the compound or its salt has an active precursor shown in formula I, and therefore can be mixed with IgG to specifically modify a specific site in the Fc region.
• a drug or labeling substance can be bound to IgG via the reactive functional group.
• an antibody-drug conjugate in which the drug is bound site-specifically can be obtained. This allows the properties of the drug to be imparted to IgG, and therefore can be applied to drug delivery systems (DDS), etc.
• DDS drug delivery systems
• the amount of the labeling substance can be controlled, which is useful for tracking, distribution, detection, etc. of IgG.
• the salt of the compound represented by formula (1) above is not particularly limited as long as it is a pharmacologically acceptable salt, and may be either an acid salt or a basic salt.
• the salt include alkali metal salts such as lithium salt, sodium salt, and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, oxalate, and phosphate, as well as acetate, propionate, hexanoate, cyclopentanepropionate, glycolate, pyruvate, lactate, malonate, succinate, malate, fumarate, tartrate, citrate, benzoate, o-(4-hydroxybenzoyl)benzoate, cinnamate, mandelate, and methanesulfonate.
• alkali metal salts such as lithium salt, sodium salt, and potassium salt
• alkaline earth metal salts such as magnesium salt and calcium
• ethanesulfonate 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, 2-naphthalenesulfonate, p-toluenesulfonate, camphorsulfonate, glucoheptanoate, 3-phenylpropionate, trimethyl acetate, tert-butyl acetate, lauryl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate, stearate, trifluoroacetate (TFA) salt, maleate, muconate, and other organic acid salts.
• TFA trifluoroacetate
• the conjugation step provides a modified antibody as shown in formula (2) below.
• (P1-S) n -Ab Formula (2) In formula (2), Ab is IgG. n is 1 or 2.
• FIG. 1 illustrates a reaction for modifying IgG with a compound in which P1 in the above formula (1) is a labeling site that can be labeled with a radioisotope.
• FIG. 1 shows a modified antibody in which n is 1. Since the compound has an active precursor, it binds to IgG via an activated compound. This reaction adds a substituent containing R2 to a specific amino acid residue of IgG, and R3 containing IgG-BP is not added to IgG. This allows IgG to be easily modified with P1 in the above formula (1).
• the compound is exposed to IgG.
• the reaction conditions in the complexation step may be, for example, mixing the compound and IgG in a buffer solution of pH 7.0 to 9.0. The concentrations of the compound and IgG in the buffer solution are adjusted as appropriate.
• the molar ratio of the compound to IgG in the buffer solution is, for example, 1:1 to 10, 1:2 to 8, or 1:3 to 6, and is preferably 1:6.
• the method for producing a radiolabeled antibody according to this embodiment may further include a labeling step of labeling the labeling site with a radioisotope.
• P1 in the above formula (1) is a labeling site that has click reaction group 1 (first click reaction group) and can be labeled with a radioisotope.
• the method for producing a radiolabeled antibody according to this embodiment may further include, prior to the labeling step, a complex formation step of reacting a compound represented by the following formula (3) with a radioisotope to form a complex of the radioisotope and a chelating agent to obtain a compound for labeling.
• Ch is a chelating agent.
• L1 is a linker having a click reactive group 2 (second click reactive group) that reacts with click reactive group 1.
• the chelating agent has a structure derived from the compound shown in formula V below or a salt thereof.
• R 5 , R 6 and R 7 are each independently -(CH 2 ) p COOH, -(CH 2 ) p C 5 H 4 N, -(CH 2 ) p PO 3 H 2 , -(CH 2 ) p CONH 2 or -(CHCOOH)(CH 2 ) p COOH.
• One of R 8 and R 9 is a hydrogen atom, a carboxyl group or a carboxyalkyl group having 2 or 3 carbon atoms, and the other is a substituent for bonding to S in the above formula (2).
• p is an integer of 0 to 3.
• R 8 is a substituent for bonding to S in the above formula (2)
• R 9 is a hydrogen atom.
• R 8 is not a substituent for bonding to S in the above formula (2)
• R 9 is a substituent for bonding to S in the above formula (2).
• the structure derived from the compound represented by the above formula V or a salt thereof is a structure in which at least one atom constituting formula V is substituted with a linker represented by L1.
• a complex between the radioisotope and the chelating agent can be formed by a known method.
• an aqueous solution containing the compound shown in formula (3) above and sodium acetate, an aqueous solution containing gentisic acid and sodium acetate, and a hydrochloric acid solution containing the radioisotope can be mixed and reacted at 50 to 90°C, 60 to 80°C, preferably 70°C, for 30 to 90 minutes, 40 to 80 minutes, preferably 60 minutes.
• the method for producing a radioactively labeled antibody includes a complexation step, it may further include a labeling step in which click reaction group 1 possessed by the modified antibody obtained in the complexation step is reacted with click reaction group 2 possessed by the labeling compound obtained in the complexation step to label the modified antibody with a radioisotope.
• Click reaction group 1 and click reaction group 2 can be reacted by a known method.
• the compound shown in formula (1) binds to IgG via an activated compound, so IgG-BP is not added to IgG. Therefore, the modified antibody obtained by the method for producing a radiolabeled antibody according to this embodiment can avoid a decrease in blood retention caused by remaining IgG-BP. Therefore, even if a bivalent modified antibody is included, the modified antibody has sufficient blood retention.
• the modified antibody is useful as a radiopharmaceutical.
• radiolabeled Antibody In another embodiment, a radiolabeled antibody is provided, the radiolabeled antibody being shown in formula (4): (P2-S)n-Ab Formula (4)
• P2 is a complex moiety that contains a radioisotope, preferably a complex formed by a radioactive metal nuclide as the radioisotope and a chelating agent.
• S is a spacer.
• the spacer (S) comprises Lys-Gly-Gly, and the amino group of the side chain of Lys in the spacer in formula (4) above is replaced with P2.
• the spacer in formula (4) above specifically binds to Lys in the Fc region of IgG.
• S in formula (4) is selected from the group consisting of a polyethylene glycol (PEG) chain, an alkylene chain (-CH 2 -), and a combination thereof.
• the degree of polymerization (p) of the PEG chain is, for example, 2 to 30, 3 to 28, 3 to 27, 3 to 25, or 3 to 24.
• the number of carbon atoms q of the alkylene chain is, for example, 1 to 10, 1 to 8, 1 to 6, or 1 to 4.
• the spacer may also contain a PEG chain and an alkylene chain.
• the degree of polymerization of (P2-S) is n
• the above formula (4) is represented by (P2-(CH 2 )q-(PEG)p)n-Ab.
• Ab is IgG.
• An antibody that specifically binds to any antigen can be used as IgG.
• antigens include proteins, sugar chains, nucleic acids, and low molecular weight compounds.
• the antigen is a protein.
• proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors, ligands, and soluble receptors, and more specifically, may be target proteins related to diseases.
• an antibody that recognizes an antigen specifically expressed in tumor cells can be used.
• antigens include PD-L1, GD2, PDGFR ⁇ (platelet-derived growth factor receptor), CD3, CD16A, CD19, CD20, CD22, CD25, CD27, CD30, CD32B, CD33, CD37, CD38, CD39, CD40, CD70, CD73, CD74, CD79b, CD100 (SEMA4D), CD105 (endoglin), CD123, CD137 (4-1BB), CD138, CD157 (Bst1), CD166, CD200, HER2, HER3, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGF, VEGFR-2, VEGF-A, HG F, gpNMB, DEC-205, folate receptor, Trop2, CEACAM5, S1P, IGF-1R, DLL4, TNT-1/B, CPAAs, PSMA, ICAM-1, MUC1, EGFR,
• n 1 or 2, preferably n is 2.
• the spacer (S) in formula (4) is linked to, for example, Lys248 or Lys246, preferably Lys248, according to the Eu numbering in human IgG Fc.
• P2 in the above formula (4) may include a group shown in the following formula (5).
• ChM is the complex moiety
• L2 is a linker.
• the linker in formula (5) may contain any one of the groups shown in formulas VI-1, VI-2, or VI-3 below.
• R 11 represents a binding site to the ChM side in formula (5).
• R 12 represents a binding site to the S side in formula (4).
• one of R 13 and R 14 is a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group, and the other represents a binding site to the ChM side in formula (5).
• R 15 represents a binding site to the S side in formula (4).
• the radiolabeled antibody represented by formula (4) is a radiolabeled antibody in which a radiolabeled linker represented by the following formula (4-1) or formula (4-2) is bound to a lysine residue of human IgG Fc (for example, Lys248 or Lys246 according to Eu numbering in human IgG Fc, preferably Lys248).
• a radiolabeled linker represented by the following formula (4-1) or formula (4-2) is bound to a lysine residue of human IgG Fc (for example, Lys248 or Lys246 according to Eu numbering in human IgG Fc, preferably Lys248).
• one or two molecules, preferably two molecules, of the radiolabeled linker shown in formula (4-1) or formula (4-2) are bound to one human IgG molecule.
• the human IgG is a human antibody represented by Ab of the radiolabeled antibody represented by formula (4).
• p is, for example, 2 to 30, 3 to 28, 3 to 27, 3 to 25, or 3 to 24.
• the wavy line indicates the binding site with the lysine residue of human IgG Fc.
• q is, for example, 1 to 10, 1 to 8, 1 to 6, or 1 to 4.
• the wavy line indicates the binding site with the lysine residue of human IgG Fc.
• human IgG or human antibody may refer to any antibody that is partially derived from a human, and includes not only fully human antibodies, but also chimeric antibodies and humanized antibodies.
• the radiolabeled antibody according to the present embodiment is used for the purpose of treating a disease, it is preferable to use an ⁇ -ray emitting nuclide or a ⁇ - ray emitting nuclide as the radioisotope from the viewpoint of enhancing the therapeutic effect.
• the ⁇ -ray emitting nuclide may be any nuclide that emits ⁇ -rays in the decay process of the radioisotope, and is preferably a radioactive metal nuclide that emits ⁇ -rays.
• the radioactive metal nuclide that emits ⁇ -rays is preferably 212 Bi, 213 Bi, 227 Th or 225 Ac, etc., more preferably 227 Th or 225 Ac, and even more preferably 225 Ac.
• the ⁇ - ray emitting nuclide may be any nuclide that emits ⁇ - rays in the decay process of a radioisotope, but is preferably a radioactive metal nuclide that emits ⁇ - rays.
• the radioactive metal nuclide that emits ⁇ - rays is preferably 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, and more preferably 64 Cu, 67 Cu, 89 Sr, 90 Y or 177 Lu is used.
• the radiolabeled antibody according to the present embodiment is used for the purpose of diagnosing a disease or detecting a lesion, it is preferable to use a ⁇ + ray emitting nuclide, an electron capture decay nuclide, or a ⁇ -ray emitting nuclide as the radioisotope in order to improve diagnostic performance.
• the ⁇ + ray emitting nuclide may be any nuclide that emits a positron in the decay process of the radioisotope, and is preferably a metal nuclide that emits a positron, such as 44Sc , 58Co , 68Ga , 64Cu , or 89Zr , and more preferably 64Cu or 89Zr .
• the electron capture decay nuclide may be any nuclide that emits Auger electrons or characteristic X-rays during the decay process of the radioisotope, and preferably used are radioactive metal nuclides such as 51 Cr, 57 Co, 58 Co, 67 Ga, 68 Ga, 64 Cu, 89 Zr, 111 In, 186 Re, 201 Tl, or 197 Hg.
• the gamma-ray emitting nuclide may be any nuclide that emits gamma rays by gamma decay, and preferably used are radioactive metal nuclides such as 99m Tc, 68 Ga, or 201 Tl.
• radioactive metal nuclides coordinated in an ionic state to the radioactive metal complex as the radioisotope are selected based on the ionic radius
• examples of radioactive metals having an ionic radius of about 70 to 130 pm include 67 Ga, 68 Ga, 64 Cu, 67 Cu, 89 Zr, 90 Y, 99m Tc, 103 Ru, 111 In, 153 Sm, 165 Dy, 166 Ho, 177 Lu, 186 Re, 188 Re, 198 Au, 201 Tl, 197 Hg, 203 Hg, 212 Bi, 213 Bi, 212 Pb, and 225 Ac.
• These radioactive metal nuclides can form a complex of the radioactive metal nuclide with a chelating agent having the structure shown in formula (4) above.
• the radioactive metal nuclide mentioned above is preferably one or more selected from Al18F, 64Cu, 67Cu, 68Ga, 89Zr, 90Y , 99mTc , 111In , 177Lu and 225Ac .
• a radiopharmaceutical in another embodiment, contains the above-mentioned radiolabeled antibody as an active ingredient.
• the radiopharmaceutical can be formulated according to a conventional method.
• the radiopharmaceutical may contain any pharmacologically acceptable component.
• the optional components include, for example, carriers, excipients, lubricants, binders, disintegrants, solvents, solubilizers, suspending agents, isotonicity agents, buffers, and soothing agents.
• additives such as preservatives, antioxidants, colorants, and sweeteners may be added to the radiolabeled antibody as necessary.
• the route of administration of the radioactive pharmaceutical is not particularly limited.
• the radioactive pharmaceutical may be administered, for example, parenterally or orally. In the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, transdermal administration, nasal administration, pulmonary administration, enteral administration, transmucosal administration, etc.
• the radioactive pharmaceutical may be administered via infusion.
• the radioactive pharmaceutical may be formulated in any form.
• the subject of administration of the radiopharmaceutical is preferably a vertebrate, more preferably a mammal.
• mammals include humans, chimpanzees and other primates, pigs and horses, as well as birds such as ducks and chickens.
• a particularly preferred subject of administration is humans.
• the radiopharmaceutical is used in internal radiotherapy of cancer or in the diagnosis of cancer.
• the radiolabeled antibody specifically binds to a target expressed in the cancer, in particular a marker molecule for the cancer.
• the cancer is not particularly limited, but examples thereof include breast cancer, brain tumor, prostate cancer, pancreatic cancer, stomach cancer, lung cancer, colon cancer, rectal cancer, large intestine cancer, small intestine cancer, esophageal cancer, duodenal cancer, tongue cancer, pharyngeal cancer, salivary gland cancer, neurilemmoma, liver cancer, kidney cancer, bile duct cancer, endometrial cancer, cervical cancer, ovarian cancer, bladder cancer, skin cancer, hemangioma, malignant lymphoma, malignant melanoma, thyroid cancer, parathyroid cancer, nasal cavity cancer, paranasal sinus cancer, bone tumor, angiofibroma, retinal sarcoma, penile cancer, testicular tumor, pediatric solid tumor, sarcoma, leukemia, and the like.
• These cancers may be primary or metastatic. IgG may be appropriately selected depending on the type of cancer.
• trastuzumab may be used for breast cancer
• P1-SY-B Formula (1) (In formula (1), P1 is a labeling moiety that is or can be labeled with a radioisotope; S is a spacer, Y is a reactive site that reacts with IgG, B is an antibody binding site that includes an IgG-binding peptide that specifically binds to the Fc region of IgG. or a salt thereof with IgG, In the formula (1), Y has a group represented by the formula I. Methods for producing radiolabeled antibodies. [2] R 1 is a nitrophenoxy group. A method for producing a radiolabeled antibody according to [1].
• the method further comprises a labeling step of labeling the labeling site with the radioisotope.
• P1 is a labeling moiety that has a first click reaction group and can be labeled with a radioisotope
• Ch-L1 formula (3) (In formula (3), Ch is a chelating agent; L1 is a linker having a second click reactive group that reacts with the first click reactive group. and reacting the compound represented by the formula (I) with the radioisotope to form a complex between the radioisotope and the chelating agent to obtain a labeling compound.
• the method further comprises a labeling step of reacting the first click reaction group of the modified antibody obtained in the conjugation step with the second click reaction group of the labeling compound obtained in the complex formation step, thereby labeling the modified antibody with the radioisotope.
• the chelating agent has a structure derived from the compound represented by formula V or a salt thereof.
• the following formula (4): (P2-S)n-Ab Formula (4) (In formula (4), P2 comprises a radioisotope; S is a spacer, Ab is IgG, n is 1 or 2. As shown in S in the formula (4) contains Lys-Gly-Gly, and the amino group of the side chain of Lys in S in the formula (4) is substituted with P2; In the formula (4), S specifically binds to Lys in the Fc region of IgG. Radiolabeled antibodies.
• P2 comprises a radioisotope; S is a spacer, Ab is IgG, n is 1 or 2. As shown in S in the formula (4) is selected from the group consisting of a polyethylene glycol chain, an alkylene chain, and a combination thereof; In the formula (4), S specifically binds to Lys in the Fc region of IgG. Radiolabeled antibodies.
• P2 is a complex moiety containing a complex formed by a radioisotope and a chelating agent; The radiolabeled antibody according to [16] or [17].
• the radioisotope is a radioactive halogen atom, Al 18 F, 64 Cu, 68 Ga, 89 Zr, 90 Y, 99 m Tc, 111 In, 177 Lu or 225 Ac;
• the chelating agent has a structure derived from the compound represented by formula V or a salt thereof.
• n is 2.
• the IgG is trastuzumab or panitumumab.
• P2 in the formula (4) is The following formula (5): ChM-L2 Equation (5) (In formula (5), ChM is the complex moiety, and L2 is a linker.) Including the group shown in The radiolabeled antibody according to [18].
• L2 in the formula (5) is Contains any one of the groups shown in formula VI-1, VI-2 or VI-3 above; The radiolabeled antibody according to [23].
• a composition comprising the radiolabeled antibody according to any one of [16] to [24] as an active ingredient. Radiopharmaceuticals. [26] Used in radiotherapy for cancer or in cancer diagnosis. The radiopharmaceutical according to [25].
• Example 1 Preparation of radioactive metal nuclide-labeled trastuzumab conjugate (Azide group linker modification of trastuzumab)
• the compound A was synthesized as follows: 0.25 mmol of Rink Amide PEG resin (0.55 mmol/g) was used, and a protected peptide resin was constructed by repeating Fmoc removal with piperidine/1-methyl-2-pyrrolidone (1:4) and Fmoc-protected amino acid/DIC/Oxyma (1 mmol/1 mmol/1 mmol) using a PurePep Chorus peptide solid-phase synthesizer (Gyros Protein Technologies).
• the obtained protected peptide resin (0.25 mmol) was reacted with 4-nitrophenyl chloroformate (0.5 g) in dichloromethane for 1 hour to construct a PMD (phenoxycarbonylated N-methyl-o-diaminobenzene) (NO 2 ) skeleton.
• the obtained resin was treated with a TFA solution to remove the resin and perform deprotection, and solidified with diethyl ether to obtain 966 mg of crude peptide (2SH form).
• the obtained crude peptide was purified with a reversed-phase HPLC (high performance liquid chromatography) column and lyophilized to obtain 147 mg of the 2SH form.
• Compound A was reacted with an antibody (human IgG1 antibody drug Herceptin, trastuzumab, manufactured by Roche).
• Trastuzumab is an antibody that uses HER2 as an antigen.
• 600 ⁇ L of 0.5 mol/L bicarbonate buffer (pH 8.9) was added to 300 ⁇ L of 20 mg/mL IgG solution, and 2075.7 ⁇ L of distilled water was added.
• 8.1 ⁇ L of 10 mmol/L compound A dissolved in dimethyl sulfoxide (DMSO) was added, and after rapid stirring, the mixture was allowed to react at room temperature for 20 minutes.
• DMSO dimethyl sulfoxide
• Figure 2 shows the elution chromatogram (280 nm) of the antibody solution.
• the peak at 1.92 minutes corresponds to the elution of compound A.
• the peak at 2.25 minutes corresponds to the elution of IgG.
• Figure 3A shows the results of mass spectrometry of the IgG peak in the antibody solution before the addition of compound A.
• Figure 3B shows the results of mass spectrometry of the IgG peak in the reaction solution.
• molecular species 1 148,221 Da
• molecular species of 148,532 Da and 148,843 Da increased.
• 89Zr -labeled DOTAGA-DBCO 89Zr obtained by the nuclear reaction of 89Y(p,n) 89Zr and prepared as an aqueous solution of 89Zr4 + ions (manufactured by Nippon Medi-Physics Co., Ltd.) was added to a vial, and the solvent was distilled off. 0.1 mol /L hydrochloric acid was added to the vial and the bottom surface was thoroughly washed by pipetting to obtain 30 ⁇ L of 0.1 mol/L hydrochloric acid containing 101.3 MBq of 89Zr4 + ions as radioactivity.
• 0.5 ⁇ L was extracted from the obtained reaction mixture of 89 Zr-labeled DOTAGA-DBCO and separated by thin layer chromatography (TLC, iTLC SG, Agilent, developing solvent: a mixture of water for injection and acetonitrile (volume ratio 1:1)).
• TLC thin layer chromatography
• the thin layer chromatogram after development was introduced into a TLC analyzer (GITAStar, MS Equipment Co., Ltd.) to measure the total 89 Zr radioactivity count including unreacted 89 Zr in the reaction mixture and the radioactivity count of 89 Zr-labeled DOTAGA-DBCO.
• the percentage of the radioactivity count of 89 Zr-labeled DOTAGA-DBCO to the total 89 Zr radioactivity count was 97.4%.
• a 89Zr -trastuzumab conjugate was obtained by a two-step process consisting of 89Zr -labeling of the ligand and a click reaction of the 89Zr -ligand. That is, 72.4 ⁇ L of solution A (containing 4.5 nmol of antibody) was added to 30 ⁇ L of solution B (containing 4.5 nmol of 89Zr-labeled DOTAGA-DBCO) and reacted at 37° C. for 90 minutes to bind the antibody and 89Zr -labeled DOTAGA-DBCO by a click reaction.
• the reaction rate of the obtained 89 Zr-trastuzumab conjugate was calculated as follows.
• the total 89 Zr radioactivity count including unreacted 89 Zr in the reaction mixture and the radioactivity count of the 89 Zr-trastuzumab conjugate were separated by TLC (iTLC SG, manufactured by Agilent, developing solvent: a mixture of an aqueous EDTA solution (0.1 mol/L) and acetonitrile (volume ratio 1:1)).
• TLC iTLC SG, manufactured by Agilent, developing solvent: a mixture of an aqueous EDTA solution (0.1 mol/L) and acetonitrile (volume ratio 1:1)
• the thin-layer chromatogram after development was measured with a TLC analyzer (GITAStar, manufactured by MS Instruments Co., Ltd.), and the percentage of the radioactivity count of the 89 Zr-trastuzumab conjugate relative to the total 89 Zr radioactivity count was 95.3%.
• trastuzumab formulation buffer solution was added to the solution obtained as described above to adjust the radioactivity concentration of the 89 Zr-trastuzumab conjugate to 10 MBq/mL.
• the total 89Zr radioactivity count including unreacted 89Zr in the purified solution and the radioactivity count of the 89Zr -trastuzumab conjugate were measured by TLC in the same manner as above, and the radiochemical purity (RCP) of the 89Zr -trastuzumab conjugate (hereinafter also referred to as "Example 1") was 97.8%.
• Comparative Example 1 Production of 89Zr -trastuzumab conjugate (monovalent) by CCAP method (Synthesis of peptide-modified trastuzumab)
• An IgG-BP containing 17 amino acid residues represented by the following formula VII was prepared by the method described in WO 2017/217347.
• the amino acid sequence of this IgG-BP is an amino acid sequence (SEQ ID NO: 153) in which X5 in SEQ ID NO: 2 is K.
• the side chain terminal amino group of the lysine residue of the IgG-BP is modified with the structure represented by R16 in the following formula VII.
• a disulfide bond is formed between two cysteine residues, and an azide group is bound to the N-terminus of the peptide via a linker structure having diglycolic acid and eight PEGs.
• the C-terminus of the IgG-BP is amidated.
• GPDCAYHKGELVWCTFH (SEQ ID NO: 153) in -GPDCAYHK(R 16 )-GELVWCTFH- represents an amino acid in one letter code.
• trastuzumab 50 mg was dissolved in 25 mL of 20 mmol/L acetic acid-sodium acetate buffer (pH 6.0) to obtain a 2 mg/mL antibody solution. Then, 233 ⁇ L (1.7 equivalents relative to the antibody) of the prepared IgG-BP DMSO solution (peptide concentration: 2.4 mmol/L) was added, followed by immediate pipetting and leaving to react at room temperature for 60 minutes to obtain a solution containing peptide-modified trastuzumab.
• the solution containing the peptide-modified trastuzumab obtained as described above was diluted with 25 mL of 20 mmol/L acetic acid-sodium acetate buffer (pH 6.0) and passed through an IgG-BP column (a column in which 5,632 nmol of the peptide whose amino acid sequence is shown in SEQ ID NO: 154 is immobilized on a 5 mL Hitrap NHS-activated HP column (manufactured by Cytiva), the peptide is cross-linked with a disulfide bond) to retain the peptide-modified trastuzumab.
• IgG-BP column a column in which 5,632 nmol of the peptide whose amino acid sequence is shown in SEQ ID NO: 154 is immobilized on a 5 mL Hitrap NHS-activated HP column (manufactured by Cytiva), the peptide is cross-linked with a disulfide bond) to retain the peptide-modified tras
• a 0.10 mol/L acetic acid-sodium acetate buffer solution (pH 5.7) containing 0.15 mol/L sodium chloride was passed through this column to remove the peptide-modified antibody in which one trastuzumab molecule was modified with two peptide molecules (hereinafter referred to as "bivalent peptide-modified trastuzumab"), and then a 0.1 mol/L glycine hydrochloride buffer solution (pH 3.5) was passed through the column to collect a fraction containing a peptide-modified trastuzumab in which one trastuzumab molecule was modified with one peptide molecule (hereinafter referred to as "monovalent peptide-modified trastuzumab").
• the entire amount of the recovered solution obtained above was added to an ultrafiltration filter (Amicon Ultra-15, Merck) to which 10 mL of trastuzumab formulation buffer had been added beforehand, and centrifuged in a centrifuge (centrifugation conditions: 25°C, 3000 x g, 20 minutes). After centrifugation, the filtrate was discarded, and 13.5 mL of trastuzumab formulation buffer was added again and centrifuged under the same conditions. This centrifugation was performed twice in total.
• a trastuzumab formulation buffer was added to the solution obtained as described above to obtain a monovalent peptide-modified trastuzumab solution (hereinafter also referred to as "Solution C") in which the concentration of monovalent peptide-modified trastuzumab was adjusted to 15 mg/mL.
• trastuzumab formulation buffer solution was added to the resulting solution to adjust the radioactivity concentration of the 89 Zr-trastuzumab conjugate (monovalent) to 10 MBq/mL.
• the RCP of the obtained 89Zr -trastuzumab conjugate (monovalent) was calculated as follows: The total 89Zr radioactivity count including unreacted 89Zr in the purified solution and the radioactivity count of the 89Zr -trastuzumab conjugate (monovalent) were measured by TLC in the same manner as in Example 1, and the RCP of the 89Zr -trastuzumab conjugate (monovalent) (hereinafter also referred to as "Comparative Example 1”) was 98.7%.
• Comparative Example 2 Preparation of 89Zr -trastuzumab conjugate (divalent) by CCAP method (Synthesis of peptide-modified trastuzumab) 10 mg of trastuzumab was dissolved in 2.5 mL of 20 mmol/L acetic acid-sodium acetate buffer (pH 6.0) to obtain a 4 mg/mL antibody solution. Then, 164 ⁇ L (6 equivalents relative to the antibody) of the DMSO solution of the IgG-BP (peptide concentration: 2.4 mmol/L) was added, followed by immediate pipetting and allowing to stand at room temperature for reaction for 60 minutes to obtain a solution containing peptide-modified trastuzumab.
• the entire solution containing peptide-modified trastuzumab was added to an ultrafiltration filter (Amicon Ultra-15, Merck) to which 10 mL of trastuzumab formulation buffer had been added beforehand, and centrifuged in a centrifuge (centrifugation conditions: 25°C, 3000 x g, 20 minutes). After centrifugation, the filtrate was discarded, and 13.5 mL of trastuzumab formulation buffer was added again and centrifuged under the same conditions. This centrifugation was performed twice in total.
• an ultrafiltration filter Amicon Ultra-15, Merck
• a bivalent peptide-modified trastuzumab solution (hereinafter also referred to as "Solution D") was obtained in which the concentration of the bivalent peptide-modified trastuzumab was adjusted to 15 mg/mL.
• the reaction rate of the obtained 89Zr -trastuzumab conjugate (bivalent) was calculated as follows: The total 89Zr radioactivity count including unreacted 89Zr in the reaction mixture of the obtained 89Zr-trastuzumab conjugate (bivalent) and the radioactivity count of the 89Zr - trastuzumab conjugate (bivalent) were evaluated in the same manner as in Example 1. As a result, the percentage of the radioactivity count of the 89Zr -trastuzumab conjugate to the total 89Zr radioactivity count was 95.4%.
• a trastuzumab formulation buffer solution was added to the solution obtained as described above to adjust the radioactivity concentration of the 89 Zr-trastuzumab conjugate (divalent) to 10 MBq/mL.
• the RCP of the 89Zr -trastuzumab conjugate (divalent) was calculated as follows: The total 89Zr radioactivity count including unreacted 89Zr in the purified solution and the radioactivity count of the 89Zr -trastuzumab conjugate (divalent) were measured by TLC in the same manner as in Example 1. As a result, the RCP of the 89Zr -trastuzumab conjugate (divalent) (hereinafter also referred to as "Comparative Example 2”) was 99.8%.
• Example 1 and Comparative Example 1 were each added to 1% bovine serum albumin-containing PBS to a concentration of 5 kBq/mL, and SK-OV-3 tumor slices and MDA-MB-231 tumor slices were immersed in the solution. The slices were contacted with an imaging plate, and then read with a scanner-type image analyzer (Typhoon FLA 7000, manufactured by GE Healthcare Japan) to evaluate the radioactivity bound to the slices.
• a scanner-type image analyzer Teleon FLA 7000, manufactured by GE Healthcare Japan
• Example 1 Comparative Example 1
• Comparative Example 2 Biodistribution Evaluation
• mice administered Example 1, Comparative Example 1, or Comparative Example 2 were kept in metabolic cages (manufactured by Shinano Seisakusho) until the time of dissection, and feces and urine were collected.
• the weights of the collected tissues and blood were measured, and the counting rates of each tissue, blood, and the collected feces and urine were measured using a gamma-ray scintillation counter (JDC-1712, Hitachi, Ltd.)
• the accumulation rate of administered radioactivity per unit weight of tissue was calculated from the obtained tissue weights and counting rates.
• FIG. 5 shows the administered radioactivity accumulation rate (mean ⁇ standard deviation) per unit weight of blood.
• Example 1 had higher accumulation in blood than Comparative Example 1 and Comparative Example 2. This indicates that Example 1 has high blood retention.
• FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, and FIG. 6E show the administered radioactivity accumulation rate (mean ⁇ standard deviation) per unit weight of the liver, kidney, femur, thigh muscle, and spleen, respectively.
• Example 1 has high accumulation in blood and increased blood retention, which is considered to increase accumulation in the whole body including the liver, kidney, femur, thigh muscle, and spleen. Therefore, it was shown that Example 1 has improved retention in the whole body.
• Example 2 Preparation of radioactive metal nuclide-labeled trastuzumab conjugate (Synthesis of azide group linker)
• the above-mentioned compound B (6-Azido hexanoic-acid), compound C (Azido-PEG3-acid) and compound D (Azido-PEG24-acid) were synthesized as follows.
• Compounds B and C were prepared using 0.3 mmol of Rink Amide PEG resin (0.5 mmol/g), and compound D was prepared using 0.15 mmol of Rink Amide PEG resin (0.5 mmol/g).
• the protected peptide resin was constructed by repeating Fmoc removal with piperidine/1-methyl-2-pyrrolidone (1:4) and condensation of amino acids with Fmoc-protected amino acid/DIC/Oxyma (1:1:1) on an ABI433A solid-phase peptide synthesizer (Applied Biosystems).
• condensation of 6-Azido hexanoic-acid was carried out with 6-Azido hexanoic-acid/EDC/HOAt (0.9 mmol/0.9 mmol/0.9 mmol)
• condensation of Azido-PEG3-acid was carried out with Azido-PEG3-acid/EDC/HOAt (0.9 mmol/0.9 mmol/0.9 mmol)
• condensation of Azido-PEG24-acid was carried out with Azido-PEG24-acid/EDC/HOAt (0.2 mmol/0.2 mmol/0.3 mmol).
• the obtained protected peptide resin was reacted with 4-nitrophenyl chloroformate (2 g/mmol) in dichloromethane for 1 hour to construct a PMD(NO 2 ) skeleton.
• the obtained resin was treated with a TFA solution to remove the resin and perform deprotection, and solidified with diethyl ether to obtain a crude peptide (2SH form).
• the obtained crude peptide was purified using a reversed-phase HPLC column and lyophilized to obtain a 2SH form. Thereafter, the crude peptide (2SH form) was dissolved in acetic acid/water (1:1), and 1 equivalent of 0.1 M iodine-methanol solution was slowly added under ice cooling.
• Tmab_N3 Synthesis of high-purity bivalent azide group linker (compound A) modified trastuzumab (hereinafter also referred to as "Tmab_N3")
• Compound A was reacted with an antibody (human IgG1 antibody drug Herceptin, trastuzumab, manufactured by Roche).
• 1000 ⁇ L of 0.5 mol/L bicarbonate buffer (pH 8.9) was added to 2310 ⁇ L of 7.8 mg/mL IgG solution, and 1629.3 ⁇ L of distilled water was added.
• 60.7 ⁇ L of 10 mmol/L compound A dissolved in DMSO was added to make the total volume 5000 ⁇ L, and after rapid stirring, the reaction was allowed to proceed at room temperature for 30 minutes.
• Tmab_6N3 Synthesis of high-purity divalent azide group linker (compound B) modified trastuzumab (hereinafter also referred to as "Tmab_6N3")
• Compound B was reacted with trastuzumab in the same manner as in Tmab_N3, except that no additional reaction was performed after dialysis (the IgG concentration in the final reaction product was 24.3 ⁇ M, and the reagent was 121.4 ⁇ mol/L at a 5-fold molar ratio).
• Figure 7A shows the results of mass spectrometry of the IgG peak in the antibody solution before the addition of compound A.
• Figure 7B shows the results of mass spectrometry of the IgG peak in the reaction solution of Tmab_N3.
• the molecular species (148,220 Da) corresponding to the molecular weight of the raw antibody decreased, and molecular species of 148,532 Da and 148,840 Da increased.
• the difference of 312 Da and 620 Da corresponded to one molecule and two molecules of azide-modified KGG (312 Da) added by the reaction of compound A, respectively. From the mass spectrum results in Figure 7B, it was confirmed that the monovalent modified form (148,532 Da) accounted for 13% and the divalent modified form (148,840 Da) accounted for 87%.
• Figure 8A shows the results of mass spectrometry of the IgG peak in the antibody solution before the addition of compound B.
• Figure 8B shows the results of mass spectrometry of the IgG peak in the reaction solution of Tmab_6N3.
• the molecular species corresponding to the molecular weight of the raw antibody 148,220 Da
• the molecular species of 148,498 Da increased.
• This difference of 278 Da coincided with the addition of two molecules of azide hexanoyl (141 Da) by the reaction of compound B.
• the mass spectrum results in Figure 8B confirmed that the divalent modification (148,498 Da) was 100%.
• Figure 9A shows the results of mass spectrometry of the IgG peak in the antibody solution before the addition of compound C.
• Figure 9B shows the results of mass spectrometry of the IgG peak in the reaction solution of Tmab_11N3.
• the molecular species corresponding to the molecular weight of the raw antibody (148,220 Da) decreased, and the molecular species of 148,650 Da increased.
• This difference of 430 Da coincided with the addition of two molecules of azide-PEG3 (217 Da) by the reaction of compound C. From the mass spectrum results in Figure 9B, it was confirmed that the monovalent modification (148,435 Da) accounted for 8% and the divalent modification (148,650 Da) accounted for 92%.
• Figure 10A shows the results of mass spectrometry of the IgG peak in the antibody solution before the addition of compound D.
• Figure 10B shows the results of mass spectrometry of the IgG peak in the reaction solution of Tmab_75N3.
• the molecular species corresponding to the molecular weight of the raw antibody (148,220 Da) decreased, and the molecular species of 150,532 Da increased.
• This difference of 2,312 Da coincided with the addition of two molecules of azide-modified PEG24 (1,156 Da) by the reaction of compound D.
• the mass spectrum results in Figure 10B confirmed that the divalent modification (150,532 Da) was 100%.
• 89Zr -labeled DOTAGA-DBCO 89 Zr obtained by the nuclear reaction of 89 Y(p,n) 89 Zr and prepared in an aqueous solution of 89 Zr 4+ ions (manufactured by Nippon Medi-Physics Co., Ltd.) was added to a vial, and the solvent was distilled off.
• 89Zr -trastuzumab conjugates were obtained by the same method as in Example 1. That is, 145 ⁇ L each of solutions E, F, G, and H (containing 14.4 nmol of antibody) was added to 100 ⁇ L of solution I (containing 12 nmol of 89Zr-labeled DOTAGA-DBCO) and reacted at 37° C. for 90 minutes to bond the antibody and 89Zr -labeled DOTAGA-DBCO by click reaction.
• 89Zr -Tmab_N3, 89Zr -Tmab_6N3, 89Zr -Tmab_11N3, and 89Zr -Tmab_75N3 were obtained from solutions E, F, G, and H, respectively, as 89Zr -trastuzumab conjugates.
• 89Zr -Tmab_N3 is a radiolabeled antibody represented by formula (4), in which S is Lys-Gly-Gly, the amino group of the side chain of Lys in S in formula (4) is substituted with P2, P2 is a group represented by formula (5), L2 is a group represented by formula VI-1, Ch is a group represented by formula V, and R 5 , R 6 , R 7 , and R 8 are each independently -CH 2 COOH, R 9 is a substituent for bonding to formula VI-1, M is 89Zr , n is 2, and Ab is trastuzumab.
• 89 Zr-Tmab — 6N3 is a radiolabeled linker represented by formula (4-2) in which q is 5 and complexed with 89 Zr, and two molecules of the linker are bound to trastuzumab.
• 889 Zr-Tmab — 11N3 is a radiolabeled linker represented by formula (4-1) in which p is 3 and complexed with 89 Zr, and two molecules of the linker are bound to trastuzumab.
• 89 Zr-Tmab — 75N3 In the radiolabeled linker represented by formula (4-1) in which 89 Zr is complexed, p is 24, and two molecules of the linker are bound to trastuzumab.
• the reaction rates of the obtained 89 Zr-trastuzumab conjugates were calculated in the same manner as in Example 1.
• the percentages of the radioactive counts of the 89 Zr-trastuzumab conjugates relative to the total 89 Zr radioactive counts were 94.8% for 89 Zr-Tmab_N3, 95.8% for 89 Zr-Tmab_6N3, 94.5% for 89 Zr-Tmab_11N3, and 82.8% for 89 Zr-Tmab_75N3.
• the conjugates were purified in the same manner as in Example 1, except that the final radioactive concentration of the 89 Zr-trastuzumab conjugates was changed to 50 MBq/mL.
• the RCP was 99.2% for 89 Zr-Tmab_N3, 99.7% for 89 Zr-Tmab_6N3, 99.5% for 89 Zr-Tmab_11N3, and 99.1% for 89 Zr-Tmab_75N3.
• Example 3 Preparation of radioactive metal nuclide-labeled panitumumab conjugate] (Synthesis of high-purity divalent azide group linker (compound A) modified panitumumab (hereinafter also referred to as "Pmab_N3”)) Compound A was reacted with an antibody (human IgG2 antibody drug Vectibix, panitumumab, manufactured by Amgen). 600 ⁇ L of 0.5 mol/L bicarbonate buffer (pH 8.9) was added to 650 ⁇ L of 20 mg/mL IgG solution, and 1705.8 ⁇ L of distilled water was added.
• an antibody human IgG2 antibody drug
• Vectibix human IgG2 antibody drug
• panitumumab manufactured by Amgen
• Solvent replacement was performed in the same manner as in Example 1, except that the formulation buffer solution was changed to a 50 mmol/L aqueous sodium acetate solution containing 0.1 mol/L sodium chloride, to obtain a Pmab_N3 solution (hereinafter also referred to as "Solution J"), a Pmab_6N3 solution (hereinafter also referred to as "Solution K”), and a Pmab_11N3 solution (hereinafter also referred to as "Solution L”) with antibody concentrations of 15 mg/mL.
• Solution J a 50 mmol/L aqueous sodium acetate solution containing 0.1 mol/L sodium chloride
• Figure 11A shows the results of mass spectrometry of the IgG peak in the antibody solution before the addition of compound A.
• Figure 11B shows the results of mass spectrometry of the IgG peak in the reaction solution of Pmab_N3.
• the molecular species corresponding to the molecular weight of the raw antibody (147,103 Da) decreased, and the molecular species of 147,710 Da increased.
• This difference of 607 Da coincided with the addition of two molecules of azido-KGG (312 Da) by the reaction of compound A.
• the mass spectrum results in Figure 11B confirmed that the divalent modification was 100%.
• Figure 12A shows the results of mass spectrometry of the IgG peak in the antibody solution before the addition of compound B.
• Figure 12B shows the results of mass spectrometry of the IgG peak in the reaction solution of Pmab_6N3.
• the molecular species corresponding to the molecular weight of the raw antibody (147,103 Da) decreased, while the molecular species of 147,373 Da increased.
• This difference of 270 Da coincided with the addition of two molecules of azide hexanoyl (141 Da) by the reaction of compound B.
• the mass spectrum results in Figure 12B confirmed that the divalent modification was 100%.
• Figure 13A shows the results of mass spectrometry of the IgG peak in the antibody solution before the addition of compound C.
• Figure 13B shows the results of mass spectrometry of the IgG peak in the reaction solution of Pmab_11N3.
• the molecular species (147,103 Da) corresponding to the molecular weight of the raw antibody decreased, and molecular species of 147,320 Da and 147,522 Da increased.
• the difference of 217 Da and 419 Da corresponded to one molecule and two molecules of azide-modified PEG3 (217 Da) added by the reaction of compound C, respectively. From the mass spectrum results in Figure 13B, it was confirmed that the monovalent modification (147,320 Da) accounted for 1% and the divalent modification (147,522 Da) accounted for 99%.
• 89Zr -labeled DOTAGA-DBCO 89 Zr obtained by the nuclear reaction of 89 Y(p,n) 89 Zr and prepared in an aqueous solution of 89 Zr 4+ ions (manufactured by Nippon Medi-Physics Co., Ltd.) was added to a vial, and the solvent was distilled off.
• 89Zr panitumumab conjugates were obtained by the same method as in Example 1. That is, 110 ⁇ L each of solutions J, K, and L (containing 10.9 nmol of antibody) was added to 100 ⁇ L of solution M (containing 9 nmol of 89Zr-labeled DOTAGA-DBCO) and reacted at 37° C. for 90 minutes to bond the antibody and 89Zr -labeled DOTAGA-DBCO by click reaction.
• 89Zr -Pmab_N3, 89Zr -Pmab_6N3, and 89Zr -Pmab_11N3 were obtained as 89Zr panitumumab conjugates from solutions J, K, and L, respectively.
• 89Zr -Pmab_N3 is a radiolabeled antibody represented by formula (4), in which S is Lys-Gly-Gly, the amino group of the side chain of Lys in S in formula (4) is substituted with P2, P2 is a group represented by formula (5), L2 is a group represented by formula VI-1, and Ch is a group represented by formula V, with the proviso that R 5 , R 6 , R 7 , and R 8 are each independently -CH 2 COOH, R 9 is a substituent for bonding to formula VI-1, M is 89Zr , n is 2, and Ab is panitumumab.
• 89 Zr-Pmab_6N3 is a compound in which q is 5 in the radiolabeled linker represented by formula (4-2) complexed with 89 Zr, and two molecules of panitumumab are bound to the linker.
• 889 Zr-Pmab — 11N3 is a compound in which two molecules of a radiolabeled linker represented by formula (4-1) in which p is 3 and complexed with 89 Zr are bound to panitumumab.
• the reaction rates of the obtained 89 Zr-panitumumab conjugates were calculated in the same manner as in Example 1.
• the percentage of the radioactive count of the 89 Zr-panitumumab conjugate relative to the total 89 Zr radioactive count was 93.3% for 89 Zr-Pmab_N3, 91.8% for 89 Zr-Pmab_6N3, and 93.5% for 89 Zr-Pmab_11N3.
• the final 89Zr -panitumumab conjugate was purified in the same manner as in Example 1, except that the radioactivity concentration was changed to 50 MBq/mL and the buffer solution was changed to a 50 mmol/L aqueous sodium acetate solution containing 0.1 mol/L sodium chloride.
• the RCP was 98.5% for 89Zr -Pmab_N3, 98.6% for 89Zr -Pmab_6N3, and 98.2% for 89Zr -Pmab_11N3.
• the entire solution containing the monovalent peptide-modified panitumumab was added to an ultrafiltration filter (Amicon Ultra-15, Merck) to which 10 mL of 50 mmol/L sodium acetate aqueous solution containing 0.1 mol/L sodium chloride had been added beforehand, and centrifuged in a centrifuge (centrifugation conditions: 25°C, 3000 x g, 20 minutes). After centrifugation, the filtrate was discarded, and 13.5 mL of 50 mmol/L sodium acetate aqueous solution containing 0.1 mol/L sodium chloride was added again, followed by centrifugation under the same conditions. This centrifugation was performed twice in total.
• a monovalent peptide-modified panitumumab solution (hereinafter also referred to as "Solution N") was obtained in which the concentration of the monovalent peptide-modified panitumumab was adjusted to 12 mg/mL.
• the reaction rate of the obtained 89 Zr panitumumab conjugate (monovalent) was calculated in the same manner as in Example 1, and the percentage of the radioactive count of the 89 Zr panitumumab conjugate relative to the total 89 Zr radioactive count was 82.6%.
• the 89Zr -labeled antibody according to the present embodiment maintained an RCP of 98% or more when stored in a refrigerator for 7 days after production, and no significant decrease in RCP was observed even after storage for 14 days.
• Sample Preparation 89Zr -Tmab_N3, 89Zr -Tmab_6N3, and 89Zr -Tmab_11N3 prepared in Example 3 were diluted to 2 mg/mL with trastuzumab formulation buffer.
• Tris aqueous solution and 10 ⁇ L of the above antibody solution were mixed in a 1.5 mL tube.
• This solution was mixed with 1 ⁇ L of 500 mmol/L DTT aqueous solution and left to stand at room temperature for 30 minutes to prepare a reduced sample (final DTT concentration: 10 mmol/L).
• the sample to which DTT was not added was prepared as a non-reduced sample.
• a Waters 2695 separation module or e2695 separation module was used as the liquid chromatography device, a Waters 2489 UV/Vis detector was used as the UV detector, and a Raytest Gabi Star detector was used as the RI detector, and the analysis was performed under the following conditions.
• Tumor volume (mm 3 ) (tumor major axis ⁇ (tumor minor axis) 2 ) ⁇ 1/2
• the obtained PET images were converted into the ratio of the radioactivity concentration of each site (Standard Uptake Value: SUV) using image analysis software PMOD (manufactured by PMOD), assuming that the administered radioactive material is uniformly distributed and not excreted, and the radioactivity concentration is set to 1.
• Volumes of interest (VOI) were set for the tumor, heart (intracardiac blood), liver, and kidneys for the SUV-converted PET images, and quantitative analysis was performed.
• Test Example 7 Antigen binding activity ( 89 Zr-labeled Pmab)] The antigen binding activity of each radioconjugate according to Example 3 and Comparative Example 3 on the day after production was confirmed by in vitro ARG.
• the evaluation was performed in the same manner as in Test Example 1, except that the tumor sections used were A431 cells, an EGFR-positive human epidermoid carcinoma-derived cell line purchased from ECACC (The European Collection of Authenticated Cell Cultures), A549 cells, an EGFR-positive human lung cancer-derived cell line purchased from ECACC, SW48 cells, an EGFR-positive colon cancer-derived cell line purchased from ATCC, HCT116 cells, an EGFR-positive human colon cancer-derived cell line purchased from ATCC, and CAMA-1 cells, an EGFR-negative human breast cancer cell line purchased from ATCC.
• the tumor sections used were A431 cells, an EGFR-positive human epidermoid carcinoma-derived cell line purchased from ECACC (The European Collection of Authenticated Cell Cultures), A549
• A431 cells an EGFR-positive cell line derived from human epidermoid carcinoma, purchased from ECACC, were suspended in PBS and administered subcutaneously to the flank of 5-week-old female BALB/c-nu/nu mice (Jackson Laboratory Japan) at 5 x 10 6 cells to generate tumor-bearing mice. Two weeks after the tumor-bearing treatment, the tumor volume was confirmed to be approximately 100 mm 3 . The tumor volume was calculated in the same manner as in Test Example 6.
• Example 4 Preparation of radioactive metal nuclide ( 225Ac )-labeled trastuzumab conjugate] (Preparation of 225Ac -labeled DOTAGA-DBCO) DOTAGA-DBCO was dispersed in 0.1 mol/L sodium acetate buffer (pH 5.0) as a solvent to obtain a dispersion containing 0.3 mmol/L of a chelating agent.
• the RCP 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 TLC (Agilent, model number: SGI0001, developing solvent: acetonitrile/water mixture (volume ratio 1:1)), and then measured by a radio ⁇ -TLC analyzer (Raytest, Model GITA Star). The percentage of the peak radioactivity (counts) detected near the origin relative to the total detected radioactivity (counts) was taken as the RCP of the 225 Ac complex. As a result, the RCP of the 225 Ac complex was 95.1%. The obtained 225 Ac complex solution was used as it is in the antibody labeling process.
• 225 Ac-Tmab_N3 is a radiolabeled antibody represented by formula (4), in which S is Lys-Gly-Gly, the amino group of the side chain of Lys in S in formula (4) is substituted with P2, P2 is a group represented by formula (5), L2 is a group represented by formula VI-1, Ch is a group represented by formula V, and R 5 , R 6 , R 7 , and R 8 are each independently -CH 2 COOH, R 9 is a substituent for bonding to formula VI-1, M is 225 Ac, n is 2, and Ab is trastuzumab.
• 225 Ac-Tmab — 6N3 is a compound in which two molecules of a radiolabeled linker represented by formula (4-2) in which q is 5 and complexed with 225 Ac are bound to trastuzumab.
• 225 Ac-Tmab — 11N3 is a compound in which two molecules of a radiolabeled linker represented by formula (4-1) in which p is 3 and complexed with 225 Ac are bound to trastuzumab.
• the amount of 225 Ac complex and the amount of azide group linker modified antibody (bivalent) were 5.7 nmol and 10.1 nmol, respectively.
• the reaction rates of the unpurified 225 Ac labeled antibodies were 41.3% (Tmab_N3), 48.0% (Tmab_6N3), and 44.6% (Tmab_11N3), respectively.
• the obtained 225 Ac labeled antibodies were purified using an ultrafiltration filter (Merck, model number: UFC505096).
• the RCPs of the purified 225 Ac labeled antibodies were 86.8% ( 225 Ac-Tmab_N3), 88.7% ( 225 Ac-Tmab_6N3), and 89.8% ( 225 Ac-Tmab_11N3), respectively.
• Comparative Example 4 Preparation of 225 Ac-Trastuzumab Conjugate (Monovalent) by CCAP Method (Antibody labeling step) Solution C obtained in Comparative Example 1 was added to the unpurified 225 Ac complex solution obtained in Example 4, and a click reaction was carried out at 37° C. for 120 minutes to obtain a 225 Ac-labeled antibody.
• the amount of the 225 Ac complex and the amount of the azide group linker-modified antibody (divalent) were 5.7 nmol and 10.1 nmol, respectively.
• the reaction rate of the unpurified 225 Ac-labeled antibody was 38.4%.
• the RCP of the purified 225 Ac-trastuzumab conjugate (monovalent) (hereinafter also referred to as "Comparative Example 4") was 93.4%.
• SK-OV-3 tumor-bearing mice were prepared in the same manner as in Test Example 6. Two weeks after the tumor-bearing treatment, the mice were randomly divided into groups from individuals with shapes suitable for tumor diameter measurement. Each radioactive complex according to Example 4 and Comparative Example 4 were administered into the tail vein at a dose of 5 kBq/mouse (15 ⁇ g/mouse as trastuzumab).
• a control group a vehicle group was set up in which a storage buffer solution was administered into the tail vein. Each group consisted of six mice, and the general condition was observed, and the body weight and tumor volume were measured over time up to 40 days after administration. In the vehicle group, due to fighting between the mice during the breeding period, some mice were subjected to a humane endpoint, and after 8 days after administration, only one mouse was kept.
• the change in tumor volume over time is shown in Figure 20.
• the vertical axis of Figure 20 shows the relative value when the tumor volume at the time of administration of each drug is set to 1.
• the horizontal axis of Figure 20 shows the number of days since administration of each drug.
• the graph shows the average tumor volume ⁇ standard deviation for each group. In the group administered with the radioactive complex, the increase in tumor volume was slower than in the vehicle group, confirming the efficacy of administration of the radioactive complex. Furthermore, each radioactive complex of Example 4 showed an antitumor effect equal to or greater than that of Comparative Example 4. There was no significant change in the general condition of each individual, and no signs of toxicity such as significant weight loss were observed.
• the present invention is useful for producing radiolabeled antibodies.

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