WO2010131590A1 - Diagnostic agent and therapeutic agent for tumors - Google Patents

Diagnostic agent and therapeutic agent for tumors Download PDF

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WO2010131590A1
WO2010131590A1 PCT/JP2010/057696 JP2010057696W WO2010131590A1 WO 2010131590 A1 WO2010131590 A1 WO 2010131590A1 JP 2010057696 W JP2010057696 W JP 2010057696W WO 2010131590 A1 WO2010131590 A1 WO 2010131590A1
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tumor
targeting molecule
patient
scfv
amino acid
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PCT/JP2010/057696
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French (fr)
Japanese (ja)
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隆雄 浜窪
嘉貴 熊倉
敏光 百瀬
浩平 津本
悟士 榎本
美穂子 宇井
俊子 先浜
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国立大学法人 東京大学
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Priority to JP2011513311A priority Critical patent/JPWO2010131590A1/en
Publication of WO2010131590A1 publication Critical patent/WO2010131590A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • 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
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A61K47/6898—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies using avidin- or biotin-conjugated 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
    • A61K51/1045—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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a tumor diagnostic agent or therapeutic agent kit comprising a combination of an antibody fragment that specifically binds to a tumor cell and a radioisotope. More specifically, the present invention relates to a tumor diagnostic agent or therapeutic agent kit characterized by defining the administration period of an antibody fragment that specifically binds to a tumor cell and a radioisotope.
  • Robo1 (roundabout, axon guidance receptor, homolog 1) is specifically expressed on the cell membrane surface of hepatocellular carcinoma, and is considered promising as a target molecule for the development of new antibody therapeutics (Non-patent Document 1) ).
  • the present inventors proceeded with the modification of anti-Robo1 IgG for micro PET imaging of nude mouse xenograft of HepG2 cells highly expressing Robo1, and VH, which is the minimum unit necessary for recognizing antigen
  • a single chain variable region fragment was constructed by connecting variable regions (Fv) composed of VL with a flexible peptide linker.
  • a combined administration method of scFv-SA and 64 Cu-DOTA-Biotin is examined for the optimization of biodistribution by pretargeting micro-PET using 64 Cu, and specifically binds to tumor cells. It was an object to be solved to provide a diagnostic or therapeutic agent kit for a tumor containing a combination of an antibody fragment and a radioisotope. Furthermore, the present invention provides a diagnostic agent for tumors, which can administer an antibody fragment that specifically binds to tumor cells and a radioisotope without using a clearing agent to diagnose or treat the tumor. Or it was set as the problem which should be solved to provide a therapeutic agent kit.
  • the present inventor has intensively studied to solve the above problems, and (a) a first targeting molecule in which an antibody fragment that specifically binds to a tumor cell and one of a pair of affinity substances are linked, And (b) tumor diagnosis or treatment using a tumor diagnostic agent or therapeutic agent kit comprising a second targeting molecule in which a radioisotope and the other of the pair of affinity substances are linked.
  • the second targeting molecule Administering the first targeting molecule to a patient in need, wherein the first targeting molecule specifically binds to a tumor site and is stably present at the tumor site, After sufficient time has passed for the first targeting molecule non-specifically adsorbed to a non-tumor site of the patient to be substantially excreted from the patient's blood, the second targeting molecule is By administering to the patient and localizing the radioisotope at the tumor site. Te, they found that tumors can be diagnosed and treated, thereby completing the present invention.
  • the following inventions are provided. (1) (a) a first targeting molecule in which an antibody fragment that specifically binds to a tumor cell and one of a pair of affinity substances are linked, and (b) a radioisotope, and the pair of A diagnostic or therapeutic kit for a tumor comprising a second targeting molecule linked to the other of the affinity substances, wherein the first targeting is performed on a patient in need of diagnosis or treatment of the tumor A molecule is administered and is non-specifically adsorbed to the non-tumor site of the patient while the first targeting molecule specifically binds to the tumor site and is stably present at the tumor site.
  • the second targeting molecule is administered to the patient and the radioisotope is A diagnostic or therapeutic agent kit for the above-mentioned tumor characterized by being localized at a tumor site G.
  • Agent kit (3) The diagnostic or therapeutic agent kit for a tumor according to (2), wherein the biotin-binding protein is avidin, streptavidin, tamavidin, or a derivative thereof.
  • the radioisotope and the other of the pair of affinity substances are linked using a chelating agent capable of binding to the radioisotope, (1) to (The diagnostic or therapeutic agent kit for a tumor according to any one of 3). (5) The tumor diagnostic or therapeutic agent kit according to any one of (1) to (4), wherein the antibody fragment that specifically binds to a tumor cell is an anti-Robo1 antibody.
  • the first targeting molecule is a tetramer or dimer molecule formed by linking a single-chain antibody fragment (scFv) that specifically binds to a tumor cell and streptavidin.
  • the tumor diagnostic or therapeutic agent kit according to any one of (1) to (5).
  • An antibody fragment that specifically binds to a tumor cell has the amino acid sequence shown in SEQ ID NO: 3 as the amino acid sequence of the heavy chain variable region, and the amino acid shown in SEQ ID NO: 4 as the amino acid sequence of the light chain variable region
  • the diagnostic or therapeutic agent kit for a tumor according to any one of (1) to (6), which is a single-chain antibody fragment (scFv) that specifically binds to Robo1, having a sequence.
  • the first targeting molecule has sufficient time for the first targeting molecule nonspecifically adsorbed to the non-tumor site of the patient to be substantially excreted from the blood of the patient.
  • the diagnostic or therapeutic agent kit for a tumor according to any one of (1) to (7), which is 6 to 144 hours after administration of.
  • the radioisotope is 64 Cu, 124 I, 76 Br, 68 Ga, 111 In, 99m Tc, 67 Ga, 123 I, 131 I, or 90 Y, (1) to (8)
  • a first targeting molecule in which an antibody fragment that specifically binds to tumor cells and one of a pair of affinity substances are linked; While the first targeting molecule specifically binds to a tumor site and is stably present at the tumor site, the first targeting molecule is nonspecifically adsorbed to a non-tumor site of the patient.
  • a second targeting molecule in which a radioisotope and the other of the pair of affinity substances are linked after a sufficient time has elapsed for the targeting molecule to be substantially excreted from the blood of the patient.
  • a method for the diagnosis or treatment of a tumor comprising administering to the patient and localizing the radioisotope to the tumor site.
  • one of the pair of affinity substances is a biotin-binding protein and the other of the pair of affinity substances is biotin or a derivative thereof.
  • the biotin-binding protein is avidin, streptavidin, tamavidin, or a derivative thereof.
  • the radioisotope and the other of the pair of affinity substances are linked using a chelating agent capable of binding to the radioisotope, from (13) to (13) The method according to any one of 15).
  • a chelating agent capable of binding to the radioisotope from (13) to (13) The method according to any one of 15).
  • the first targeting molecule is a tetramer or dimer molecule formed by linking a single-chain antibody fragment (scFv) that specifically binds to a tumor cell and streptavidin.
  • scFv single-chain antibody fragment
  • An antibody fragment that specifically binds to a tumor cell has the amino acid sequence set forth in SEQ ID NO: 3 as the amino acid sequence of the heavy chain variable region, and the amino acid set forth in SEQ ID NO: 4 as the amino acid sequence of the light chain variable region
  • the first targeting molecule has sufficient time for the first targeting molecule non-specifically adsorbed to the non-tumor site of the patient to be substantially excreted from the blood of the patient.
  • the method according to any one of (13) to (19), which is 6 hours to 144 hours after administration.
  • the radioisotope is 64 Cu, 124 I, 76 Br, 68 Ga, 111 In, 99m Tc, 67 Ga, 123 I, 131 I, or 90 Y, (13) to (20) The method in any one.
  • Second targeting of (a) an antibody fragment that specifically binds to a tumor cell and one of a pair of affinity substances for the production of a tumor diagnostic or therapeutic agent kit Use of a second targeting molecule linking a molecule and (b) a radioisotope and the other of the pair of affinity substances to a patient in need of tumor diagnosis or treatment, A first targeting molecule is administered, while the first targeting molecule specifically binds to a tumor site and is stably present at the tumor site, wherein the first targeting molecule is non- After a sufficient amount of time has elapsed for the first specifically-adsorbed molecule to be substantially excreted from the patient's blood, the second targeting molecule is administered to the patient; Use as described above, characterized in that a radioisotope is localized at the tumor site.
  • the radioisotope and the other of the pair of affinity substances are linked using a chelating agent capable of binding to the radioisotope, from (22) to ( 24) Use according to any of the above.
  • a chelating agent capable of binding to the radioisotope from (22) to ( 24) Use according to any of the above.
  • the first targeting molecule is a tetramer or dimer molecule formed by linking a single-chain antibody fragment (scFv) that specifically binds to a tumor cell and streptavidin.
  • scFv single-chain antibody fragment
  • the antibody fragment that specifically binds to a tumor cell has the amino acid sequence set forth in SEQ ID NO: 3 as the amino acid sequence of the heavy chain variable region, and the amino acid set forth in SEQ ID NO: 4 as the amino acid sequence of the light chain variable region
  • the first targeting molecule has sufficient time for the first targeting molecule non-specifically adsorbed to the non-tumor site of the patient to be substantially excreted from the patient's blood.
  • the radioisotope is 64 Cu, 124 I, 76 Br, 68 Ga, 111 In, 99m Tc, 67 Ga, 123 I, 131 I, or 90 Y, (22) to (29) Use as described in any of the above.
  • a tumor can be diagnosed or treated without using a clearing agent by using a combination of an antibody fragment that specifically binds to a tumor cell and a radioisotope.
  • FIG. 1 shows the structure of an expression vector of B5209B mouse-scFv- Streptavidin (SA).
  • FIG. 2 shows the result of final purification of B5209B scFv-Streptavidin by size exclusion chromatography.
  • FIG. 3 shows the results of isothermal titration calorimetry (ITC) of B5209B scFv-Streptavidin and ROBO1.
  • FIG. 4 shows the results of isothermal titration calorimetry (ITC) of B5209B scFv-Streptavidin and Biotin.
  • FIG. 5 shows the results of differential scanning calorimetry (DSC) of B5209B scFv-Streptavidin.
  • FIG. 1 shows the structure of an expression vector of B5209B mouse-scFv- Streptavidin (SA).
  • FIG. 2 shows the result of final purification of B5209B scFv-Streptavidin by size exclusion chromatography.
  • FIG. 6 shows the structure of a recombinant anti-ROBO1 monoclonal antibody used in micro PET imaging in the examples.
  • scFv-StreptAvdin (170 kDa including scFv-SA, SA tetramer) was prepared as a modified monoclonal antibody.
  • scFv-SA has one 64 Cu-DOTA-Biotin for each subunit of SA that forms a tetramer. Only combine.
  • FIG. 7 shows the results when 64 Cu-DOTA-Biotin and scFv-SA were mixed and administered (upper left A: 2 hours later, upper right B: one day later, lower left C: two days later, lower right D: 3 days after).
  • 64 Cu-DOTA-Biotin-SA-scFv complex has high retention in blood (A). Most of it is excreted via the liver, and because it is a large molecule, it is presumed that its transferability to the tumor is low (B, C). Although the amount of tumor transfer is small, specific binding to the tumor can be confirmed when compared with normal soft tissue (D).
  • FIG. 1 shows the results when 64 Cu-DOTA-Biotin and scFv-SA were mixed and administered (upper left A: 2 hours later, upper right B: one day later, lower left C: two days later, lower right D: 3 days after).
  • 64 Cu-DOTA-Biotin-SA-scFv complex has high retention in blood (A). Most of it is excreted via the liver
  • FIG. 8 shows the results when only 64 Cu-DOTA-Biotin was administered (no modified antibody was administered) (left A: 30 minutes later, center B: 2 hours later, right C: 1 day later). 30 minutes after administration, RI already excreted in the bladder, RI accumulated in the renal pelvis (bilateral central part of the kidney), RI accumulated in the renal parenchyma, and RI considered to have accumulated in the liver parenchyma (A). After 2 hours, 64 Cu-DOTA-Biotin is almost excreted from the renal urinary system, but accumulation in the liver continues (B). One day later, in addition to the RI image thought to have been excreted into the intestinal tract via the biliary system, a new accumulation in the tumor can be identified.
  • FIG. 9 shows an example (image one day after 64 Cu) when biotinylated asialocinci was used as a clearing agent (CA) in a pretargeting experiment.
  • the scFv-SA was administered at 400 ⁇ g 36 hours before the administration of 64 Cu-DOTA-Biotin, and the clearing agent was administered at 1000 ⁇ g 12 hours before the administration.
  • the accumulation of RI in the tumor is low, and CA has decreased in this example, and the usefulness of using CA is not observed.
  • FIG. 10 shows the results when no clearing agent (CA) was used in the pretargeting experiment (left A: 1 day after, middle B: 2 days after, right C: 3 days after).
  • FIG. 10 shows the results when no clearing agent (CA) was used in the pretargeting experiment (left A: 1 day after, middle B: 2 days after, right C: 3 days after).
  • FIG. 11 shows the results of evaluating the effect on tumor accumulation by changing the interval from scFv-SA administration to 64 Cu-DOTA-Biotin administration.
  • FIG. 12 shows a tumor growth curve for 30 days after scFv-SA 400 ⁇ g was administered to HepG2 tumor-bearing mice from the tail vein and 90 Y-labeled DOTA-Biotin 1.5 mCi was administered 4 days later.
  • FIG. 13 shows the results of measurement of scFv-Streptavidin blood concentration by ELISA.
  • FIG. 14 shows a logarithmic approximation curve of the measurement result of scFv-Streptavidin blood concentration.
  • Antibody fragment that specifically binds to tumor cells In the present invention, a first targeting molecule in which an antibody fragment that specifically binds to tumor cells and one of a pair of affinity substances are linked is used. .
  • an antibody that specifically binds to a tumor cell in addition to an antibody against Robo1 (roundabout, axon guidance receptor, homolog 1), for example, antibodies against the following tumor antigens can also be used.
  • Robo1 roundabout, axon guidance receptor, homolog 1
  • a single chain antibody fragment is preferably used, but other antibody fragments (for example, Fab, Fab ′, F (ab ′) 2, Fv, etc.) can be used.
  • the first targeting molecule is preferably a tetramer or dimer molecule comprising a single-chain antibody fragment (scFv) that specifically binds to tumor cells and streptavidin linked.
  • Preparation of antibodies against known antigens can be performed by methods known to those skilled in the art.
  • any tumor antigen or fragment thereof as an immunogen
  • any animal known to produce antibody sputum is used.
  • Immunization can be achieved by subcutaneous or intraperitoneal injection.
  • Adjuvants may be used during immunization and such adjuvants are known to those skilled in the art.
  • Monoclonal antibody sputum can be obtained by excising spleen cells from an immunized animal and fusing them with myeloma cells to produce hybridoma cells that produce monoclonal antibody sputum.
  • Hybridoma cells can be selected. A hybridoma that secretes the desired antibody sputum is cloned, cultured under appropriate conditions, and the secreted antibody is recovered and purified using methods well known in the art such as ion exchange columns, affinity chromatography, and the like. be able to.
  • DNA encoding monoclonal antibody is easily isolated and sequenced by conventional methods (e.g., using oligonucleotide probes that can specifically bind to the genes encoding heavy and light chains of monoclonal antibody) it can.
  • Hybridoma cells are a preferred starting material for such DNA.
  • a single-chain antibody fragment (scFv) having an amino acid sequence and specifically binding to Robo1 can be mentioned.
  • Radioisotope the 2nd targeting molecule which connected the radioisotope and the other of a pair of affinity substance is used.
  • Specific examples of the radioisotope that can be used in the present invention are given below.
  • Radioisotopes for treatment include beta-ray nuclides ( 32 P, 67 Cu, 89 Sr, 90 Y, 114 m In, 117 m Sn, 131 I, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, etc. ), Alpha-ray nuclides (such as 211 At, 212 Bi, 212 Pb, 213 Bi, 223 Ra, and 225 Ac) and Auger electron nuclides (such as 125 I and 165 Er) can be used.
  • beta-ray nuclides 32 P, 67 Cu, 89 Sr, 90 Y, 114 m In, 117 m Sn, 131 I, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, etc.
  • Radioisotopes for imaging include gamma ray nuclides ( 67 Ga, 99m Tc, 111 In, and 123 I), positron emitting nuclides ( 18 F, 62 Cu, 64 Cu, 66 Ga, 68 Ga, 76 Br) 86 Y, 89 Zr, 94 Tc, and 124 I) can be used.
  • positron emitting nuclides 18 F, 62 Cu, 64 Cu, 66 Ga, 68 Ga, 76 Br
  • 64 Cu, 124 I, 76 Br, 68 Ga, 111 In, 99m Tc, 123 I, 131 I, or 90 Y can be preferably used.
  • a chelating agent capable of binding to a radioisotope is used to link the radioisotope with the other of the pair of affinity substances.
  • the chelating agent that can be used in the present invention include DOTA (1,4,7,10-tetraazacyclododecane-N, N ′, N ′′, N ′ ′′-tetraacetic acid), TETA (1,4,8, 11-tetraazaryclotetradecane-N, N ′, N ′′, N ′ ′′-tetraacetic acid), N2S2, MAG3, CHX-A-DTPA, and the like.
  • a pair of affinity substances includes (a) a combination of a biotin-binding protein such as avidin, streptavidin, tamavidin, or a derivative thereof, and (b) biotin or a derivative thereof. Can do. As long as avidin, streptavidin or tamavidin and biotin have an affinity for each other, their respective derivatives can also be used.
  • the arrangement of tamavidin is described in Japanese Patent No. 4257119, JP 2008-200038, and WO 2009/028625.
  • the first targeting molecule is administered to a patient in need of tumor diagnosis or treatment, and the first targeting molecule is specific to the tumor site. And the first targeting molecule adsorbed non-specifically to the non-tumor site of the patient is substantially excreted from the blood of the patient. After sufficient time has passed, the second targeting molecule is administered to the patient, and the radioisotope is localized at the tumor site.
  • the time sufficient for the first targeting molecule nonspecifically adsorbed to the non-tumor site of the patient to be substantially excreted from the patient's blood is not particularly limited, but generally the first targeting molecule is not limited. 6 hours to 30 days after administration of one targeting molecule, preferably 6 hours to 20 days, more preferably 6 hours to 16 days, for example, 6 hours to 144 hours, 12 hours From 96 hours, or from 24 hours to 96 hours. Further, the number of administrations of the first targeting molecule is not particularly limited, and it may be administered only once, or may be administered at any number of 2 times or more (for example, 2 to 10 times). . In order to accumulate a sufficient amount of the first targeting molecule in the tumor, the first targeting molecule is preferably administered two or more times.
  • the second targeting molecule linked to the other of them may be used as a diagnostic or therapeutic agent for tumors by mixing, dissolving, emulsifying, etc. together with a pharmaceutically acceptable carrier.
  • the first targeting molecule and the second targeting molecule can be used together with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc., injectable solutions, suspensions And can be formulated into a dosage form such as an emulsion.
  • the first targeting molecule and the second targeting molecule are in an aqueous solution, preferably a physiologically compatible buffer such as Hanks's solution, Ringer's solution, or physiological saline buffer. Can be dissolved.
  • the composition can take the form of a suspension, solution, emulsion, or the like in an oily or aqueous vehicle.
  • the route of administration of the first targeting molecule and the second targeting molecule is not particularly limited, but is usually parenteral administration, such as injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.) It can be administered through the skin or transmucosal membrane.
  • parenteral administration such as injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.) It can be administered through the skin or transmucosal membrane.
  • the dosage of the first targeting molecule and the second targeting molecule is in the range of about 0.1 mg to 1000 mg, preferably about 0.1 mg per kg body weight per dose as the active ingredient dosage. To 100 mg can be administered.
  • Example 1 Preparation of modified monoclonal antibody (1) Preparation of total RNA from hybridoma cells] As a hybridoma cell producing monoclonal antibody B5209B (IgG2b), a hybridoma producing monoclonal antibody B5209B described in JP-A-2008-290996 was used.
  • the hybridoma producing this monoclonal antibody B5209B has the accession number FERM P-21238 on March 2, 2007 (National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, East of Tsukuba City, Ibaraki Prefecture, Japan).
  • the above monoclonal antibody B5209B (IgG2b) -producing 1 ⁇ 10 7 hybridoma cells were washed once with phosphate-buffered saline (PBS), and solubilized by adding 1 ml of Trizol solution (Invitrogen) to the cell precipitate. After the DNA was sheared by passing the extract through a 20 G injection needle twice, total RNA was purified by chloroform extraction, isopropanol precipitation, and 80% ethanol washing according to the instructions attached to the Trizol solution. Dissolved. It was confirmed that the obtained total RNA was not degraded by agarose gel electrophoresis.
  • PBS phosphate-buffered saline
  • MuIgVH5′-A primer of Novagen Mouse Ig-Primer Set was added to the obtained 1st strand cDNA, and double-stranded cDNA was amplified using Expand High Fidelity PCR System (Roche Diagnostics).
  • the obtained double-stranded cDNA was subcloned into pGEM-T vector (Promega) by the TA cloning method and introduced into E. coli DH5 ⁇ to obtain a plasmid-containing clone.
  • Plasmid DNA of 6 clones was purified with Qiagen Plasmid Midi Kit (manufactured by Qiagen), and the DNA base sequence was determined according to a conventional method.
  • the amino acid sequence of the heavy chain variable region (VH) of the antibody was found to be the amino acid sequence from the 1st to the 122nd amino acid sequence (amino acid sequence of SEQ ID NO: 3) of the amino acid sequence shown in SEQ ID NO: 2.
  • the amino acid sequence of the heavy chain variable region (VH) of the antibody corresponds to the amino acid sequence from the 1st to the 122nd amino acid sequence (amino acid sequence of SEQ ID NO: 3) of the amino acid sequence shown in SEQ ID NO: 2, and the light chain of the antibody
  • the amino acid sequence of the variable region (VL) corresponds to the amino acid sequence from the 142nd to the 248th amino acid sequence (amino acid sequence of SEQ ID NO: 4) of the amino acid sequence described in SEQ ID NO: 2.
  • the column used was HiLoad TM 26/60 Superdex 200 (GE healthcare) and the mobile phase was 50 mM Tris-HCl, 200 mM NaCl, pH 8.0 buffer.
  • the final purified product was confirmed by SDS-PAGE. The results of final purification by size exclusion chromatography and SDS-PAGE are shown in FIG.
  • Example 2 Evaluation of B5209B scFv-Streptavidin activity (1) Binding activity with ROBO1 Thermodynamic analysis on the interaction between B5209B scFv-Streptavidin and ROBO1 was performed by isothermal titration calorimetry (ITC).
  • FIG. 3 shows the measurement results when ROBO1 (34.7 ⁇ M) was added dropwise to B5209B scFv-Streptavidin (3.7 ⁇ M) under a condition of 25 ° C. using PBS as a solvent.
  • the dissociation constant calculated from this was 5.6 ⁇ 10 ⁇ 8 (1 / M), the enthalpy change ( ⁇ H) was ⁇ 41.4 kJ / mol, and the entropy change ( ⁇ S) was 0.52 J / mol ⁇ K.
  • ⁇ H did not change significantly.
  • ⁇ S decreased by about 1/40, and the affinity decreased by about an order of magnitude.
  • the binding ratio suggests that two molecules of ROBO1 bind to the B5209B scFv-Streptavidin tetramer, and two adjacent antigen-binding sites in the tetramer may be able to recognize only one ROBO1 due to steric hindrance. It is thought that there is sex.
  • FIG. 4 shows the measurement results when Biotin (90 ⁇ M) was added dropwise to B5209B scFv-Streptavidin (9 ⁇ M) under a condition of 25 ° C. using PBS as a solvent.
  • the binding constant calculated from this was 6.94 ⁇ 10 -9 (1 / M), the enthalpy change ( ⁇ H) was -122.2 kJ / mol, and the entropy change ( ⁇ S) was 0.25 kJ / mol ⁇ K. .
  • Example 3 Measurement of blood concentration of scFv-Streptavidin (1) Preparation of mouse serum sample 400 ⁇ g of scFv-Streptavidin was administered to cancer-bearing nude mice transplanted with HepG2 cells. After 15, 30 minutes, 1, 2, 4, 8 and 24 hours later, 10 ⁇ L of blood is collected from the tail and mixed with a sample buffer in which 3.2% sodium citrate (wako) and 1.6% Block Ace (Snow Brand Milk Products) are dissolved in PBS. A serum sample was obtained.
  • the scFv-Streptavidin concentration in the serum sample was calculated by preparing a calibration curve from a dilution series of purified Streptavidin (pierce). The results are shown in FIG.
  • the horizontal axis represents the time after scFvSA administration, and the vertical axis represents the concentration at each time in% when the scFvSA concentration 15 minutes after administration is taken as 100%.
  • Example 4 Micro PET (1) Material and method modified monoclonal antibody: From the anti-Robo1 IgG B5209 mAb (150 kDa) prepared in Example 1, scFv-StreptAvdin (scFv-SA, 170 kDa including SA tetramer) was used (FIG. 6). ScFv is 28 kDa. For any monoclonal antibody (mAb), it was confirmed in advance that the antibody binding activity was not lowered, and 400 to 1000 ⁇ g per mouse was used in the microPET experiment.
  • mAb monoclonal antibody
  • mice used and pretreatment 10 7 HepG2 cells were transplanted into 6-week-old BALB / cAjcl-nu / nu male mice, and a xenograft nude mouse model was prepared so that the body weight was about 25 g at 12 weeks of age in the microPET experiment. The size of the mass is 8-10 mm. All mice were fasted for 6 hours before positron radionuclide administration and only drinking water. In the case of the pretargeting experiment, a low biotin diet was fed about one week before the microPET experiment for the purpose of reducing endogenous Biotin.
  • Radiopharmaceutical synthesis The positron nuclide 64 Cu (half-life 12.7 hours) was produced by a method that causes a 64 Ni (p, n) 64 Cu nuclear reaction in a cyclotron. Next, 64 Cu with high specific activity was prepared in advance via the bifunctional chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Used for labeling cold DOTA-Biotin for lettergetting.
  • DOTA-Biotin 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
  • Radiopharmaceutical 64 Cu-DOTA-Biotin in the case of pretargeting was administered from a line placed in the nude mouse tail vein, and emission data was collected.
  • a clearing agent was administered for the purpose of removing scFv about a day before the scheduled time of 64 Cu administration and subsequently removing excess blood scFv at a certain time before 64 Cu administration. Specifically, we are studying multiple dosing patterns from about 3 minutes to 12 hours ago. Asialosinchi bulk powder (galactosyl human serum albumin manufactured by Japan Mediphysix) before 99m Tc labeling was obtained as a Clearing Agent, biotinylated, and about 100-1000 ⁇ g per mouse was used.
  • Image reconstruction and image data analysis (outline): The collected PET data was reconstructed into a 3D or 4D image (in the case of dynamic data collection) by the Filtered Back Projection method at the final stage after a series of processes for Study 1, Study 2, Study 3, and Study 4 and later. Image display and organ / tumor region of interest (ROI) settings were made using AsiPro, a microPET software.
  • ROI tumor region of interest
  • FIG. 9 shows an example of a nude mouse administered with 400 ⁇ g of scFv-SA 36 hours before administration of 64 Cu-DOTA-Biotin and 1000 ⁇ g of clearing agent 12 hours before administration.
  • the accumulation of RI in the tumor is low, rather the CA has decreased in this example, and the usefulness of using CA is not observed.
  • the administration method of the clearing agent we examined multiple cases where the interval between administrations of 64 Cu-DOTA-Biotin was within one hour, to 12 hours, and the CA dose was increased or decreased. It was not possible to obtain a result in which the accumulation of was better than other normal organs.
  • the interval from scFv-SA administration to 64 Cu-DOTA-Biotin administration was changed to evaluate the effect on tumor accumulation (FIG. 11).
  • the accumulation in the tumor is maintained for every 4 days, 10 days, and 16 days, and scFv-SA is not removed from the cell surface or removed (or not internalized) for a relatively long period of time.
  • FIG. 12 shows a tumor growth curve for 30 days after 90 Y-DOTA-Biotin administration.
  • the Y axis shows the tumor volume ratio when the tumor volume on the administration day is 1, and the X axis shows the time (day) after administration.
  • the tumor growth curve of the administered mice is lower than the approximate growth curve of the control mice (indicated by bold line: only 400 ⁇ g of scFv-SA and no 90 Y-DOTA-Biotin is administered), and tumor growth is controlled Controlled compared to the group.
  • the health of the administered mice was relatively good. This suggests that the pretargeting method without a clearing agent can suppress tumor growth to some extent without causing serious side effects.
  • mAbs monoclonal antibodies
  • the long retention of IgG in the blood is due to the high distribution in normal tissues.
  • the most radiosensitive hematopoietic exposure damage is fatal. Therefore, the introduction of modified mAbs is essential in developing molecular probes for imaging and treatment of solid tumors.
  • the basic kinetics of the modified mAb such as biodistribution are optimized to minimize adverse effects on normal organs and maximize the effects of imaging / treatment on the tumor site. It must be.
  • the modification of the antibody was advanced based on the mAb molecule B5209 IgG targeting Robo1, which is highly expressed in hepatocellular carcinoma, to obtain scFv which is a functional minimum fragment.
  • scFv which is a functional minimum fragment.
  • a pretargeting method in nude mice with HepG2 tumors.
  • Goldenberg et al. In the case of pretargeting using the Biotin-SA system, the use of a clearing agent is the standard to reduce excess antibodies in the blood that are not bound to cancer cell surface antigens. It has become.
  • a clearing agent containing biotin as a component is not necessarily beneficial for micro PET imaging using 64 Cu-DOTA-Biotin.
  • the biotin-binding site in scFv-SA on tumor cells may occupy a small amount of biotin derived from the clearing agent.
  • Tumor accumulation need not depend solely on specific antigen-antibody binding, and for RIT purposes it can be from any passive mechanism, such as tumor accumulation shown with 64 Cu-DOTA-Biotin alone . From the Study4 biodistribution, it is clear that hematopoietic exposure was significantly lower than whole IgG.
  • RIT In actual RIT, it is important to minimize exposure to normal organs and to obtain a tumor growth suppression / reduction effect.
  • this RIT example it was suggested that RIT by a pretargeting method without using a clearing agent with an anti-ROBO1 modified antibody was performed, and tumor growth could be suppressed to some extent without causing serious side effects.
  • RIT In order to achieve a high anti-tumor effect in pre-targeted RIT, it is natural that high accumulation of nuclides in the tumor is necessary.
  • One method is to increase the dose of the nuclide, and the upper limit of the dose of 90 Y-DOTA-Biotin is determined mainly from the viewpoint of radiation damage to the whole body.
  • a boosting method in which high accumulation in the tumor is achieved by administering a modified antibody in an amount that does not affect the living body several times at intervals is conceivable.
  • a prerequisite for this boost method is that the modified antibody must continue to accumulate on the tumor surface for some length of time.
  • scFv-SA was not removed from or removed from the cell surface over a relatively long period of time, because the accumulation in the tumor was maintained for every 4 days, 10 days, and 16 days. From this, it was shown that scFv-SA can be highly integrated by boost administration. It is expected that a higher antitumor effect can be obtained by combining this boost administration method and RIT.

Abstract

Disclosed is a diagnostic or therapeutic agent kit for tumors, which comprises a combination of an antibody fragment capable of binding specifically to tumor cells and a radioactive isotope. Specifically, the diagnostic or therapeutic agent kit for tumors comprises (a) a first targeting molecule prepared by linking an antibody fragment capable of binding specifically to tumor cells to one of a pair of affinity substances and (b) a second targeting molecule prepared by linking a radioactive isotope to the other of the pair of affinity substances. The diagnostic or therapeutic agent kit is characterized in that the first targeting molecule is administered to a patient and the second targeting molecule is administered to the patient after a lapse of a predetermined time period after the administration of the first targeting molecule.

Description

腫瘍の診断剤又は治療剤Diagnostic or therapeutic agent for tumor
　本発明は、腫瘍細胞に特異的に結合する抗体断片と放射性同位元素とを組み合わせて含む、腫瘍の診断剤又は治療剤キットに関する。より詳細には、本発明は、腫瘍細胞に特異的に結合する抗体断片と放射性同位元素との投与時期を規定することを特徴とする、腫瘍の診断剤又は治療剤キットに関する。 The present invention relates to a tumor diagnostic agent or therapeutic agent kit comprising a combination of an antibody fragment that specifically binds to a tumor cell and a radioisotope. More specifically, the present invention relates to a tumor diagnostic agent or therapeutic agent kit characterized by defining the administration period of an antibody fragment that specifically binds to a tumor cell and a radioisotope.
　Robo1(roundabout, axon guidance receptor, homolog 1)は肝細胞癌の細胞膜表面において特異的に発現が亢進しており、新しい抗体治療薬開発のための標的分子として有望視されている(非特許文献１)。本発明者らは、Robo1を高発現しているHepG2細胞のnude mouse xenograftのマイクロPET イメージングのために、anti-Robo1 IgGの改変を進め、抗原を認識するために必要な最小単位であるVHおよびVLから構成される可変領域(Fv)をフレキシブルなペプチドリンカーで結合した単鎖可変領域フラグメント(single chain Fv)を作成した。さらにscFvをストレプトアビジン(SA)化、64Cu-DOTAをビオチン化することによって、ストレプトアビジン-ビオチン結合を利用したプレターゲッティング法の導入を試みてきた。しかし，抗体の腫瘍へのターゲティングの後に投与する低分子核種が，循環血中に残存する余剰抗体により吸収され，目的の腫瘍への集積度が低下するという問題点を残していた。固形腫瘍の64Cuによるイメージングでは プレターゲッティング法を実施するために、改変抗体、除去剤（clearing agent）、および核種を順次投与することが必須であるとされている(非特許文献２)。しかし、実際には各々の投与量や投与間隔といったプレターゲッティング法の実施手順には無数とも言える組み合わせが可能である。例えば、仮に生体に順次投与する3つの物質の血液中動態が既知であったとしても、生体内の各臓器および腫瘍内で起こる複雑なinteractionや経時的変化のため、正常組織集積に比して腫瘍集積を最大とするようなプレターゲッティング法の実施手順を個々の物質に関する単独データのみから推定することは困難である。 Robo1 (roundabout, axon guidance receptor, homolog 1) is specifically expressed on the cell membrane surface of hepatocellular carcinoma, and is considered promising as a target molecule for the development of new antibody therapeutics (Non-patent Document 1) ). The present inventors proceeded with the modification of anti-Robo1 IgG for micro PET imaging of nude mouse xenograft of HepG2 cells highly expressing Robo1, and VH, which is the minimum unit necessary for recognizing antigen A single chain variable region fragment (single chain Fv) was constructed by connecting variable regions (Fv) composed of VL with a flexible peptide linker. Furthermore, we have tried to introduce a pretargeting method using a streptavidin-biotin bond by converting scFv to streptavidin (SA) and 64 Cu-DOTA to biotinylation. However, the low molecular weight nuclide administered after targeting of the antibody to the tumor is absorbed by the surplus antibody remaining in the circulating blood, and the degree of accumulation in the target tumor remains. In imaging of solid tumors with 64 Cu, it is considered essential to sequentially administer a modified antibody, a clearing agent, and a nuclide in order to carry out the prelettering method (Non-patent Document 2). However, in practice, there can be countless combinations of the pretargeting methods such as each dose and administration interval. For example, even if the kinetics in the blood of three substances that are sequentially administered to a living body are known, the complex interactions and changes over time that occur in each organ and tumor in the living body, compared to normal tissue accumulation. It is difficult to estimate the pre-targeting procedure to maximize tumor accumulation from only single data on individual substances.
　本発明においては、64Cuを用いたプレターゲッティング マイクロPET によって、biodistributionの最適化のために、scFv-SA及び64Cu-DOTA-Biotinの組み合わせ投与方法を検討し、腫瘍細胞に特異的に結合する抗体断片と放射性同位元素とを組み合わせて含む腫瘍の診断剤又は治療剤キットを提供することを解決すべき課題とした。さらに本発明は、除去剤（clearing agent）を使用することなく、腫瘍細胞に特異的に結合する抗体断片と放射性同位元素とを投与して腫瘍を診断又は治療することができる、腫瘍の診断剤又は治療剤キットを提供することを解決すべき課題とした。 In the present invention, a combined administration method of scFv-SA and 64 Cu-DOTA-Biotin is examined for the optimization of biodistribution by pretargeting micro-PET using 64 Cu, and specifically binds to tumor cells. It was an object to be solved to provide a diagnostic or therapeutic agent kit for a tumor containing a combination of an antibody fragment and a radioisotope. Furthermore, the present invention provides a diagnostic agent for tumors, which can administer an antibody fragment that specifically binds to tumor cells and a radioisotope without using a clearing agent to diagnose or treat the tumor. Or it was set as the problem which should be solved to provide a therapeutic agent kit.
　本発明者は上記課題を解決するために鋭意検討し、（ａ）腫瘍細胞に特異的に結合する抗体断片と、一対の親和性物質のうちの一方とを連結した第一の標的化分子、及び（ｂ）放射性同位元素と、前記一対の親和性物質のうちの他方とを連結した第二の標的化分子を含む、腫瘍の診断剤又は治療剤キットを用いて、腫瘍の診断又は治療を必要とする患者に、前記第一の標的化分子を投与し、前記第一の標的化分子が腫瘍部位に特異的に結合し、かつ当該腫瘍部位に安定して存在する間であって、前記患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間が経過した後に、前記第二の標的化分子を前記患者に投与し、前記放射性同位元素を前記腫瘍部位に局在化させることによって、腫瘍を診断及び治療できることを見出し、本発明を完成するに至った。 The present inventor has intensively studied to solve the above problems, and (a) a first targeting molecule in which an antibody fragment that specifically binds to a tumor cell and one of a pair of affinity substances are linked, And (b) tumor diagnosis or treatment using a tumor diagnostic agent or therapeutic agent kit comprising a second targeting molecule in which a radioisotope and the other of the pair of affinity substances are linked. Administering the first targeting molecule to a patient in need, wherein the first targeting molecule specifically binds to a tumor site and is stably present at the tumor site, After sufficient time has passed for the first targeting molecule non-specifically adsorbed to a non-tumor site of the patient to be substantially excreted from the patient's blood, the second targeting molecule is By administering to the patient and localizing the radioisotope at the tumor site. Te, they found that tumors can be diagnosed and treated, thereby completing the present invention.
　即ち、本発明によれば、以下の発明が提供される。
（１）　（ａ）腫瘍細胞に特異的に結合する抗体断片と、一対の親和性物質のうちの一方とを連結した第一の標的化分子、及び（ｂ）放射性同位元素と、前記一対の親和性物質のうちの他方とを連結した第二の標的化分子を含む、腫瘍の診断剤又は治療剤キットであって、腫瘍の診断又は治療を必要とする患者に、前記第一の標的化分子を投与し、前記第一の標的化分子が腫瘍部位に特異的に結合し、かつ当該腫瘍部位に安定して存在する間であって、前記患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間が経過した後に、前記第二の標的化分子を前記患者に投与し、前記放射性同位元素を前記腫瘍部位に局在化させることを特徴とする、上記の腫瘍の診断剤又は治療剤キット。
（２）　一対の親和性物質のうちの一方が、ビオチン結合性タンパク質であり、一対の親和性物質のうちの他方がビオチン又はその誘導体である、（１）に記載の腫瘍の診断剤又は治療剤キット。
（３）　ビオチン結合性タンパク質が、アビジン、ストレプトアビジン、タマビジン、又はそれらの誘導体である、（２）に記載の腫瘍の診断剤又は治療剤キット。 That is, according to the present invention, the following inventions are provided.
(1) (a) a first targeting molecule in which an antibody fragment that specifically binds to a tumor cell and one of a pair of affinity substances are linked, and (b) a radioisotope, and the pair of A diagnostic or therapeutic kit for a tumor comprising a second targeting molecule linked to the other of the affinity substances, wherein the first targeting is performed on a patient in need of diagnosis or treatment of the tumor A molecule is administered and is non-specifically adsorbed to the non-tumor site of the patient while the first targeting molecule specifically binds to the tumor site and is stably present at the tumor site. After sufficient time has passed for the first targeting molecule to be substantially excreted from the patient's blood, the second targeting molecule is administered to the patient and the radioisotope is A diagnostic or therapeutic agent kit for the above-mentioned tumor characterized by being localized at a tumor site G.
(2) The tumor diagnostic agent or treatment according to (1), wherein one of the pair of affinity substances is a biotin-binding protein and the other of the pair of affinity substances is biotin or a derivative thereof. Agent kit.
(3) The diagnostic or therapeutic agent kit for a tumor according to (2), wherein the biotin-binding protein is avidin, streptavidin, tamavidin, or a derivative thereof.
（４）　第二の標的化分子において、放射性同位元素と結合しうるキレート剤を用いて、放射性同位元素と前記一対の親和性物質のうちの他方とが連結している、（１）から（３）の何れかに記載の腫瘍の診断剤又は治療剤キット。
（５）　腫瘍細胞に特異的に結合する抗体断片が、抗Ｒｏｂｏ１抗体である、（１）から（４）の何れかに記載の腫瘍の診断剤又は治療剤キット。 (4) In the second targeting molecule, the radioisotope and the other of the pair of affinity substances are linked using a chelating agent capable of binding to the radioisotope, (1) to ( The diagnostic or therapeutic agent kit for a tumor according to any one of 3).
(5) The tumor diagnostic or therapeutic agent kit according to any one of (1) to (4), wherein the antibody fragment that specifically binds to a tumor cell is an anti-Robo1 antibody.
（６）　第一の標的化分子が、腫瘍細胞に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）と、ストレプトアビジとを連結してなる４量体又は２量体の分子である、（１）から（５）の何れかに記載の腫瘍の診断剤又は治療剤キット。
（７）　腫瘍細胞に特異的に結合する抗体断片が、重鎖可変領域のアミノ酸配列として配列番号３に記載のアミノ酸配列を有し、軽鎖可変領域のアミノ酸配列として配列番号４に記載のアミノ酸配列を有する、Ｒｏｂｏ１に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）である、（１）から（６）の何れかに記載の腫瘍の診断剤又は治療剤キット。 (6) The first targeting molecule is a tetramer or dimer molecule formed by linking a single-chain antibody fragment (scFv) that specifically binds to a tumor cell and streptavidin. The tumor diagnostic or therapeutic agent kit according to any one of (1) to (5).
(7) An antibody fragment that specifically binds to a tumor cell has the amino acid sequence shown in SEQ ID NO: 3 as the amino acid sequence of the heavy chain variable region, and the amino acid shown in SEQ ID NO: 4 as the amino acid sequence of the light chain variable region The diagnostic or therapeutic agent kit for a tumor according to any one of (1) to (6), which is a single-chain antibody fragment (scFv) that specifically binds to Robo1, having a sequence.
（８）　前記患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間が、前記第一の標的化分子の投与後６時間から１４４時間である、（１）から（７）の何れかに記載の腫瘍の診断剤又は治療剤キット。
（９）　前記放射性同位元素が、64Ｃｕ、124Ｉ、76Ｂｒ、68Ｇａ、111Ｉｎ、99mＴｃ、67Ｇａ、123Ｉ、131Ｉ、または90Ｙである、（１）から（８）の何れかに記載の腫瘍の診断剤又は治療剤キット。 (8) The first targeting molecule has sufficient time for the first targeting molecule nonspecifically adsorbed to the non-tumor site of the patient to be substantially excreted from the blood of the patient. The diagnostic or therapeutic agent kit for a tumor according to any one of (1) to (7), which is 6 to 144 hours after administration of.
(9) The radioisotope is 64 Cu, 124 I, 76 Br, 68 Ga, 111 In, 99m Tc, 67 Ga, 123 I, 131 I, or 90 Y, (1) to (8) A diagnostic or therapeutic agent kit for a tumor according to any one of the above.
（１０）　重鎖可変領域のアミノ酸配列として配列番号３に記載のアミノ酸配列を有し、軽鎖可変領域のアミノ酸配列として配列番号４に記載のアミノ酸配列を有する、Ｒｏｂｏ１に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）。
（１１）　一対の親和性物質のうちの一方が連結されている、（１０）に記載の一本鎖抗体フラグメント（ｓｃＦｖ）。
（１２）　（１０）又は（１１）に記載の一本鎖抗体フラグメント（ｓｃＦｖ）をコードする核酸。 (10) One that specifically binds to Robo1 having the amino acid sequence of SEQ ID NO: 3 as the amino acid sequence of the heavy chain variable region and the amino acid sequence of SEQ ID NO: 4 as the amino acid sequence of the light chain variable region Single chain antibody fragment (scFv).
(11) The single chain antibody fragment (scFv) according to (10), wherein one of a pair of affinity substances is linked.
(12) A nucleic acid encoding the single-chain antibody fragment (scFv) according to (10) or (11).
（１３）　腫瘍の診断又は治療を必要とする患者に、腫瘍細胞に特異的に結合する抗体断片と、一対の親和性物質のうちの一方とを連結した第一の標的化分子を投与し、前記第一の標的化分子が腫瘍部位に特異的に結合し、かつ当該腫瘍部位に安定して存在する間であって、前記患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間が経過した後に、放射性同位元素と、前記一対の親和性物質のうちの他方とを連結した第二の標的化分子を前記患者に投与し、前記放射性同位元素を前記腫瘍部位に局在化させることを含む、腫瘍の診断又は治療のための方法。
（１４）　一対の親和性物質のうちの一方が、ビオチン結合性タンパク質であり、一対の親和性物質のうちの他方がビオチン又はその誘導体である、（１３）に記載の方法。
（１５）　ビオチン結合性タンパク質が、アビジン、ストレプトアビジン、タマビジン、又はそれらの誘導体である、（１４）に記載の方法。 (13) administering to a patient in need of tumor diagnosis or treatment a first targeting molecule in which an antibody fragment that specifically binds to tumor cells and one of a pair of affinity substances are linked; While the first targeting molecule specifically binds to a tumor site and is stably present at the tumor site, the first targeting molecule is nonspecifically adsorbed to a non-tumor site of the patient. A second targeting molecule in which a radioisotope and the other of the pair of affinity substances are linked after a sufficient time has elapsed for the targeting molecule to be substantially excreted from the blood of the patient. A method for the diagnosis or treatment of a tumor comprising administering to the patient and localizing the radioisotope to the tumor site.
(14) The method according to (13), wherein one of the pair of affinity substances is a biotin-binding protein and the other of the pair of affinity substances is biotin or a derivative thereof.
(15) The method according to (14), wherein the biotin-binding protein is avidin, streptavidin, tamavidin, or a derivative thereof.
（１６）　第二の標的化分子において、放射性同位元素と結合しうるキレート剤を用いて、放射性同位元素と前記一対の親和性物質のうちの他方とが連結している、（１３）から（１５）の何れかに記載の方法。
（１７）　腫瘍細胞に特異的に結合する抗体断片が、抗Ｒｏｂｏ１抗体である、（１３）から（１６）の何れかに記載の方法。 (16) In the second targeting molecule, the radioisotope and the other of the pair of affinity substances are linked using a chelating agent capable of binding to the radioisotope, from (13) to (13) The method according to any one of 15).
(17) The method according to any one of (13) to (16), wherein the antibody fragment that specifically binds to a tumor cell is an anti-Robo1 antibody.
（１８）　第一の標的化分子が、腫瘍細胞に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）と、ストレプトアビジとを連結してなる４量体又は２量体の分子である、（１３）から（１７）の何れかに記載の方法。
（１９）　腫瘍細胞に特異的に結合する抗体断片が、重鎖可変領域のアミノ酸配列として配列番号３に記載のアミノ酸配列を有し、軽鎖可変領域のアミノ酸配列として配列番号４に記載のアミノ酸配列を有する、Ｒｏｂｏ１に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）である、（１３）から（１８）の何れかに記載の方法。 (18) The first targeting molecule is a tetramer or dimer molecule formed by linking a single-chain antibody fragment (scFv) that specifically binds to a tumor cell and streptavidin. (13) The method according to any one of (17).
(19) An antibody fragment that specifically binds to a tumor cell has the amino acid sequence set forth in SEQ ID NO: 3 as the amino acid sequence of the heavy chain variable region, and the amino acid set forth in SEQ ID NO: 4 as the amino acid sequence of the light chain variable region The method according to any one of (13) to (18), which is a single-chain antibody fragment (scFv) that specifically binds to Robo1 having a sequence.
（２０）　前記患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間が、前記第一の標的化分子の投与後６時間から１４４時間である、（１３）から（１９）の何れかに記載の方法。
（２１）　前記放射性同位元素が、64Ｃｕ、124Ｉ、76Ｂｒ、68Ｇａ、111Ｉｎ、99mＴｃ、67Ｇａ、123Ｉ、131Ｉ、または90Ｙである、（１３）から（２０）の何れかに記載の方法。 (20) The first targeting molecule has sufficient time for the first targeting molecule non-specifically adsorbed to the non-tumor site of the patient to be substantially excreted from the blood of the patient. The method according to any one of (13) to (19), which is 6 hours to 144 hours after administration.
(21) The radioisotope is 64 Cu, 124 I, 76 Br, 68 Ga, 111 In, 99m Tc, 67 Ga, 123 I, 131 I, or 90 Y, (13) to (20) The method in any one.
（２２）　腫瘍の診断剤又は治療剤キットの製造のための、（ａ）腫瘍細胞に特異的に結合する抗体断片と、一対の親和性物質のうちの一方とを連結した第一の標的化分子、及び（ｂ）放射性同位元素と、前記一対の親和性物質のうちの他方とを連結した第二の標的化分子の使用であって、腫瘍の診断又は治療を必要とする患者に、前記第一の標的化分子を投与し、前記第一の標的化分子が腫瘍部位に特異的に結合し、かつ当該腫瘍部位に安定して存在する間であって、前記患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間が経過した後に、前記第二の標的化分子を前記患者に投与し、前記放射性同位元素を前記腫瘍部位に局在化させることを特徴とする、上記の使用。
（２３）　一対の親和性物質のうちの一方が、ビオチン結合性タンパク質であり、一対の親和性物質のうちの他方がビオチン又はその誘導体である、（２２）に記載の使用。
（２４）　ビオチン結合性タンパク質が、アビジン、ストレプトアビジン、タマビジン、又はそれらの誘導体である、（２３）に記載の使用。 (22) First targeting of (a) an antibody fragment that specifically binds to a tumor cell and one of a pair of affinity substances for the production of a tumor diagnostic or therapeutic agent kit Use of a second targeting molecule linking a molecule and (b) a radioisotope and the other of the pair of affinity substances to a patient in need of tumor diagnosis or treatment, A first targeting molecule is administered, while the first targeting molecule specifically binds to a tumor site and is stably present at the tumor site, wherein the first targeting molecule is non- After a sufficient amount of time has elapsed for the first specifically-adsorbed molecule to be substantially excreted from the patient's blood, the second targeting molecule is administered to the patient; Use as described above, characterized in that a radioisotope is localized at the tumor site.
(23) The use according to (22), wherein one of the pair of affinity substances is a biotin-binding protein and the other of the pair of affinity substances is biotin or a derivative thereof.
(24) The use according to (23), wherein the biotin-binding protein is avidin, streptavidin, tamavidin, or a derivative thereof.
（２５）　第二の標的化分子において、放射性同位元素と結合しうるキレート剤を用いて、放射性同位元素と前記一対の親和性物質のうちの他方とが連結している、（２２）から（２４）の何れかに記載の使用。
（２６）　腫瘍細胞に特異的に結合する抗体断片が、抗Ｒｏｂｏ１抗体である、（２２）から（２５）の何れかに記載の使用。 (25) In the second targeting molecule, the radioisotope and the other of the pair of affinity substances are linked using a chelating agent capable of binding to the radioisotope, from (22) to ( 24) Use according to any of the above.
(26) The use according to any one of (22) to (25), wherein the antibody fragment that specifically binds to a tumor cell is an anti-Robo1 antibody.
（２７）　第一の標的化分子が、腫瘍細胞に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）と、ストレプトアビジとを連結してなる４量体又は２量体の分子である、（２２）から（２６）の何れかに記載の使用。
（２８）　腫瘍細胞に特異的に結合する抗体断片が、重鎖可変領域のアミノ酸配列として配列番号３に記載のアミノ酸配列を有し、軽鎖可変領域のアミノ酸配列として配列番号４に記載のアミノ酸配列を有する、Ｒｏｂｏ１に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）である、（２２）から（２７）の何れかに記載の使用。 (27) The first targeting molecule is a tetramer or dimer molecule formed by linking a single-chain antibody fragment (scFv) that specifically binds to a tumor cell and streptavidin. (22) Use in any one of (26).
(28) The antibody fragment that specifically binds to a tumor cell has the amino acid sequence set forth in SEQ ID NO: 3 as the amino acid sequence of the heavy chain variable region, and the amino acid set forth in SEQ ID NO: 4 as the amino acid sequence of the light chain variable region The use according to any one of (22) to (27), which is a single-chain antibody fragment (scFv) that specifically binds to Robo1, having a sequence.
（２９）　前記患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間が、前記第一の標的化分子の投与後６時間から１４４時間である、（２２）から（２８）の何れかに記載の使用。
（３０）　前記放射性同位元素が、64Ｃｕ、124Ｉ、76Ｂｒ、68Ｇａ、111Ｉｎ、99mＴｃ、67Ｇａ、123Ｉ、131Ｉ、または90Ｙである、（２２）から（２９）の何れかに記載の使用。 (29) The first targeting molecule has sufficient time for the first targeting molecule non-specifically adsorbed to the non-tumor site of the patient to be substantially excreted from the patient's blood. The use according to any one of (22) to (28), which is 6 hours to 144 hours after administration of.
(30) The radioisotope is 64 Cu, 124 I, 76 Br, 68 Ga, 111 In, 99m Tc, 67 Ga, 123 I, 131 I, or 90 Y, (22) to (29) Use as described in any of the above.
　本発明によれば、腫瘍細胞に特異的に結合する抗体断片と放射性同位元素とを組み合わせて用いることによって、除去剤（clearing agent）を使用することなく腫瘍を診断又は治療することができる。 According to the present invention, a tumor can be diagnosed or treated without using a clearing agent by using a combination of an antibody fragment that specifically binds to a tumor cell and a radioisotope.
図１は、B5209B mouse-scFv- Streptavidin（SA）の発現ベクターの構造を示す。FIG. 1 shows the structure of an expression vector of B5209B mouse-scFv- Streptavidin (SA). 図２は、サイズ排除クロマトグラフィーによりB5209B scFv-Streptavidinを最終精製した結果を示す。FIG. 2 shows the result of final purification of B5209B scFv-Streptavidin by size exclusion chromatography. 図３は、B5209B scFv-StreptavidinとROBO1の等温滴定型熱量測定(ITC)の結果を示す。FIG. 3 shows the results of isothermal titration calorimetry (ITC) of B5209B scFv-Streptavidin and ROBO1. 図４は、B5209B scFv-StreptavidinとBiotinの等温滴定型熱量測定(ITC)の結果を示す。FIG. 4 shows the results of isothermal titration calorimetry (ITC) of B5209B scFv-Streptavidin and Biotin. 図５は、 B5209B scFv-Streptavidinの示差走査型熱量測定(DSC)の結果を示す。FIG. 5 shows the results of differential scanning calorimetry (DSC) of B5209B scFv-Streptavidin. 図６は、実施例におけるマイクロPETイメージングで使用した遺伝子組み換え抗ROBO1モノクローナル抗体の構造を示す。抗Robo1 whole IgG B5209 mAb (150kDa)をもとに、改変モノクローナル抗体としてscFv-StreptAvdin (scFv-SA, SA四量体を含め170kDa)を作成した。なお、IgGのFc部分には、複数のDOTAが導入される余地があるのに対し、scFv-SAでは四量体を形成するSAのそれぞれのsubunitに対して、一つの64Cu-DOTA-Biotinしか結合しない。FIG. 6 shows the structure of a recombinant anti-ROBO1 monoclonal antibody used in micro PET imaging in the examples. Based on anti-Robo1 whole IgG B5209 mAb (150 kDa), scFv-StreptAvdin (170 kDa including scFv-SA, SA tetramer) was prepared as a modified monoclonal antibody. There is room for introduction of multiple DOTAs in the Fc part of IgG, whereas scFv-SA has one 64 Cu-DOTA-Biotin for each subunit of SA that forms a tetramer. Only combine. 図７は、64Cu-DOTA-BiotinとscFv-SAを混和して投与した場合の結果を示す（左上A: 2時間後、右上B: 1日後、左下C: 2日後、右下D: 3日後)。64Cu-DOTA-Biotin-SA-scFv複合体の血中滞留性は高い(A)。ほとんどが肝臓経由で排泄され、大分子のため腫瘍への移行性は低いと推察される(B, C)。腫瘍への移行は量的には少ないが、正常軟部組織と比べた場合には腫瘍への特異的結合を確認することができる(D)。FIG. 7 shows the results when 64 Cu-DOTA-Biotin and scFv-SA were mixed and administered (upper left A: 2 hours later, upper right B: one day later, lower left C: two days later, lower right D: 3 days after). 64 Cu-DOTA-Biotin-SA-scFv complex has high retention in blood (A). Most of it is excreted via the liver, and because it is a large molecule, it is presumed that its transferability to the tumor is low (B, C). Although the amount of tumor transfer is small, specific binding to the tumor can be confirmed when compared with normal soft tissue (D). 図８は、64Cu-DOTA-Biotinのみを投与した場合（改変抗体は投与していない）の結果を示す（左A: 30分後、中央B: 2時間後、右C: 1日後）。投与後30分では膀胱へすでに排泄されたRI、腎盂に貯留しているRI（両側腎臓中央部）、腎実質に集積しているRI、および肝実質に集積していると考えられるRIが描出されている(A)。2時間後では腎尿路系から64Cu-DOTA-Biotinはほとんど排泄されているにもかかわらず、肝臓への集積が持続している(B)。一日後では、胆道系を介して腸管へ排泄されたと考えられるRI像に加えて、新たに腫瘍への集積が同定できる。肝臓への集積は依然として最も高い(C)。FIG. 8 shows the results when only 64 Cu-DOTA-Biotin was administered (no modified antibody was administered) (left A: 30 minutes later, center B: 2 hours later, right C: 1 day later). 30 minutes after administration, RI already excreted in the bladder, RI accumulated in the renal pelvis (bilateral central part of the kidney), RI accumulated in the renal parenchyma, and RI considered to have accumulated in the liver parenchyma (A). After 2 hours, 64 Cu-DOTA-Biotin is almost excreted from the renal urinary system, but accumulation in the liver continues (B). One day later, in addition to the RI image thought to have been excreted into the intestinal tract via the biliary system, a new accumulation in the tumor can be identified. Accumulation in the liver is still the highest (C). 図９は、プレターゲッティング実験において、ビオチン化アシアロシンチをclearing agent(CA)として用いた場合の一例(64Cuから一日後の画像)を示す。64Cu-DOTA-Biotinの投与36時間前にscFv-SAを400μg投与し、12時間前にclearing agentを1000μg投与した。腫瘍へのRIの集積度は低く、CAはこの例では集積度を下げており、CA使用の有用性は認められない。FIG. 9 shows an example (image one day after 64 Cu) when biotinylated asialocinci was used as a clearing agent (CA) in a pretargeting experiment. The scFv-SA was administered at 400 μg 36 hours before the administration of 64 Cu-DOTA-Biotin, and the clearing agent was administered at 1000 μg 12 hours before the administration. The accumulation of RI in the tumor is low, and CA has decreased in this example, and the usefulness of using CA is not observed. 図１０は、プレターゲッティング実験において、clearing agent(CA)を使用しなかった場合（左A: 1日後、中央B: 2日後、右C: 3日後）の結果を示す。FIG. 10 shows the results when no clearing agent (CA) was used in the pretargeting experiment (left A: 1 day after, middle B: 2 days after, right C: 3 days after). 図１１は、scFv-SA投与から64Cu-DOTA-Biotin投与までの間隔を変化させて、腫瘍集積への影響を評価した結果を示す。FIG. 11 shows the results of evaluating the effect on tumor accumulation by changing the interval from scFv-SA administration to 64 Cu-DOTA-Biotin administration. 図１２は、HepG2担癌マウスに、scFv-SA400μgを尾静脈より投与し、その４日後に90Yを標識したDOTA-Biotin  1.5mCiを投与した後30日間の腫瘍増殖曲線を示す。FIG. 12 shows a tumor growth curve for 30 days after scFv-SA 400 μg was administered to HepG2 tumor-bearing mice from the tail vein and 90 Y-labeled DOTA-Biotin 1.5 mCi was administered 4 days later. 図１３は、ELISAによるscFv-Streptavidin血中濃度の測定の結果を示す。FIG. 13 shows the results of measurement of scFv-Streptavidin blood concentration by ELISA. 図１４は、scFv-Streptavidin血中濃度の測定結果の対数近似曲線を示す。FIG. 14 shows a logarithmic approximation curve of the measurement result of scFv-Streptavidin blood concentration.
　以下、本発明について更に詳細に説明する。
（１）腫瘍細胞に特異的に結合する抗体断片
　本発明では、腫瘍細胞に特異的に結合する抗体断片と、一対の親和性物質のうちの一方とを連結した第一の標的化分子を用いる。 Hereinafter, the present invention will be described in more detail.
(1) Antibody fragment that specifically binds to tumor cells In the present invention, a first targeting molecule in which an antibody fragment that specifically binds to tumor cells and one of a pair of affinity substances are linked is used. .
　腫瘍細胞に特異的に結合する抗体としては、Ｒｏｂｏ１(roundabout, axon guidance receptor, homolog 1)に対する抗体のほか、例えば、以下の腫瘍抗原に対する抗体を使用することもできる。 As an antibody that specifically binds to a tumor cell, in addition to an antibody against Robo1 (roundabout, axon guidance receptor, homolog 1), for example, antibodies against the following tumor antigens can also be used.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　抗体断片としては、一本鎖抗体フラグメント（ｓｃＦｖ）を用いることが好ましいが、それ以外の抗体断片（例えば、Fab、Fab'、F(ab')2、Fvなど）を用いることができる。
　第一の標的化分子は好ましくは、腫瘍細胞に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）と、ストレプトアビジンとを連結してなる４量体又は２量体の分子である。 As the antibody fragment, a single chain antibody fragment (scFv) is preferably used, but other antibody fragments (for example, Fab, Fab ′, F (ab ′) 2, Fv, etc.) can be used.
The first targeting molecule is preferably a tetramer or dimer molecule comprising a single-chain antibody fragment (scFv) that specifically binds to tumor cells and streptavidin linked.
　既知の抗原に対する抗体の作成は、当業者に公知の方法により行うことができる。腫瘍細胞に特異的に結合する抗体を作成するためには、まず、腫瘍抗原またはそのフラグメントを免疫原として用いて、抗体 を生成することが知られている任意の動物（マウス、ウサギ等）に皮下または腹膜内注射することにより免疫することができる。免疫に際してアジュバントを用いてもよく、そのようなアジュバントは当業者に公知である。モノクローナル抗体 は、免疫した動物から脾臓細胞を切除し、ミエローマ細胞と融合させ、モノクローナル抗体 を産生するハイブリドーマ細胞を作製することにより得ることができる。ＥＬＩＳＡアッセイ、ウエスタンブロット分析、ラジオイムノアッセイ、細胞表面上に腫瘍抗原を発現している細胞を用いたＦＡＣＳ等の当該技術分野においてよく知られる方法を用いて、当該腫瘍抗原を認識する抗体 を産生するハイブリドーマ細胞を選択することができる。所望の抗体 を分泌するハイブリドーマをクローニングし、適切な条件下で培養し、分泌された抗体を回収し、当該技術分野においてよく知られる方法、例えばイオン交換カラム、アフィニティークロマトグラフィー等を用いて精製することができる。 Preparation of antibodies against known antigens can be performed by methods known to those skilled in the art. In order to create an antibody that specifically binds to a tumor cell, first, using any tumor antigen or fragment thereof as an immunogen, any animal (mouse, rabbit, etc.) known to produce antibody sputum is used. Immunization can be achieved by subcutaneous or intraperitoneal injection. Adjuvants may be used during immunization and such adjuvants are known to those skilled in the art. Monoclonal antibody sputum can be obtained by excising spleen cells from an immunized animal and fusing them with myeloma cells to produce hybridoma cells that produce monoclonal antibody sputum. Using well-known methods in the art such as ELISA assay, Western blot analysis, radioimmunoassay, FACS using cells expressing tumor antigen on the cell surface, antibody antibody that recognizes the tumor antigen is produced. Hybridoma cells can be selected. A hybridoma that secretes the desired antibody sputum is cloned, cultured under appropriate conditions, and the secreted antibody is recovered and purified using methods well known in the art such as ion exchange columns, affinity chromatography, and the like. be able to.
　モノクローナル抗体 をコードするDNAは、慣用な方法(例えば、モノクローナル抗体 の重鎖および軽鎖をコードする遺伝子に特異的に結合することができるオリゴヌクレオチドプローブを用いて)により容易に単離、配列決定できる。ハイブリドーマ細胞はこのようなDNAの好ましい出発材料である。一度単離したDNA（例えば、モノクローナル抗体の重鎖をコードする遺伝子、およびモノクローナル抗体の軽鎖をコードする遺伝子）を発現ベクターに挿入し、E.coli細胞、COS細胞、CHO細胞または形質転換されなければ免疫グロブリンを産生しないミエローマ細胞等の宿主細胞へ組換え、組換え宿主細胞からモノクローナル抗体を産生させることができる。 DNA encoding monoclonal antibody is easily isolated and sequenced by conventional methods (e.g., using oligonucleotide probes that can specifically bind to the genes encoding heavy and light chains of monoclonal antibody) it can. Hybridoma cells are a preferred starting material for such DNA. Once isolated DNA (eg, gene encoding heavy chain of monoclonal antibody and gene encoding light chain of monoclonal antibody) is inserted into an expression vector and E. coli cells, COS cells, CHO cells or transformed Otherwise, recombination into host cells such as myeloma cells that do not produce immunoglobulins, and monoclonal antibodies can be produced from the recombinant host cells.
　本発明で用いることができる抗Ｒｏｂｏ１抗体の具体例としては、重鎖可変領域のアミノ酸配列として配列番号３に記載のアミノ酸配列を有し、軽鎖可変領域のアミノ酸配列として配列番号４に記載のアミノ酸配列を有する、Ｒｏｂｏ１に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）を挙げることができる。 Specific examples of the anti-Robo1 antibody that can be used in the present invention include the amino acid sequence of SEQ ID NO: 3 as the amino acid sequence of the heavy chain variable region, and the amino acid sequence of the light chain variable region described in SEQ ID NO: 4. A single-chain antibody fragment (scFv) having an amino acid sequence and specifically binding to Robo1 can be mentioned.
（２）放射性同位元素
　本発明では、放射性同位元素と、一対の親和性物質のうちの他方とを連結した第二の標的化分子を用いる。本発明で用いることができる放射性同位元素の具体例を以下に挙げる。、治療用の放射性同位元素としては、ベータ線核種（32P、67Cu、89Sr、90Y、114mIn、117mSn、131I、153Sm、166Ho、177Lu、186Re 、188Re など）、アルファ線核種（211At、212Bi、212Pb、213Bi、223Ra及び225Acなど）、オージェ電子核種（125I、及び165Erなど）を用いることができる。また、イメージング用の放射性同位元素としては、ガンマ線核種（67Ga、99mTc、111In、及び123Iなど）、ポジトロン放出核種（18F、62Cu、64Cu、66Ga、68Ga、76Br、86Y、89Zr、94Tc、及び124Iなど）を用いることができる。上記の中でも、好ましくは、64Ｃｕ、124Ｉ、76Ｂｒ、68Ｇａ、111Ｉｎ、99mＴｃ、123Ｉ、131Ｉ、または90Ｙを用いることができる。 (2) Radioisotope In this invention, the 2nd targeting molecule which connected the radioisotope and the other of a pair of affinity substance is used. Specific examples of the radioisotope that can be used in the present invention are given below. , Radioisotopes for treatment include beta-ray nuclides ( 32 P, 67 Cu, 89 Sr, 90 Y, 114 m In, 117 m Sn, 131 I, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, etc. ), Alpha-ray nuclides (such as 211 At, 212 Bi, 212 Pb, 213 Bi, 223 Ra, and 225 Ac) and Auger electron nuclides (such as 125 I and 165 Er) can be used. Radioisotopes for imaging include gamma ray nuclides ( 67 Ga, 99m Tc, 111 In, and 123 I), positron emitting nuclides ( 18 F, 62 Cu, 64 Cu, 66 Ga, 68 Ga, 76 Br) 86 Y, 89 Zr, 94 Tc, and 124 I) can be used. Among the above, 64 Cu, 124 I, 76 Br, 68 Ga, 111 In, 99m Tc, 123 I, 131 I, or 90 Y can be preferably used.
（３）キレート剤
　本発明における第二の標的化分子においては、放射性同位元素と結合しうるキレート剤を用いて、放射性同位元素を、前記一対の親和性物質のうちの他方とを連結することができる。本発明で用いることができるキレート剤としては、ＤＯＴＡ（1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid）、ＴＥＴＡ（1,4,8,11-tetraazaryclotetradecane-N,N',N'',N'''-tetraacetic acid）、Ｎ２Ｓ２、ＭＡＧ３、ＣＨＸ－Ａ－ＤＴＰＡなどを挙げることができる。 (3) Chelating agent In the second targeting molecule in the present invention, a chelating agent capable of binding to a radioisotope is used to link the radioisotope with the other of the pair of affinity substances. Can do. Examples of the chelating agent that can be used in the present invention include DOTA (1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N ′ ″-tetraacetic acid), TETA (1,4,8, 11-tetraazaryclotetradecane-N, N ′, N ″, N ′ ″-tetraacetic acid), N2S2, MAG3, CHX-A-DTPA, and the like.
（４）一対の親和性物質
　一対の親和性物質としては、（ａ）アビジン、ストレプトアビジン、タマビジン、又はそれらの誘導体等のビオチン結合性タンパク質と、（ｂ）ビオチン又はその誘導体の組み合わせを挙げることができる。アビジン、ストレプトアビジン又はタマビジンと、ビオチンとは、互いに親和性を有する限りは、それぞれの誘導体を使用することもできる。なお、タマビジンの配列は、特許第4257119号、特開2008-200038、ＷＯ２００９／０２８６２５に記載されている。 (4) A pair of affinity substances A pair of affinity substances includes (a) a combination of a biotin-binding protein such as avidin, streptavidin, tamavidin, or a derivative thereof, and (b) biotin or a derivative thereof. Can do. As long as avidin, streptavidin or tamavidin and biotin have an affinity for each other, their respective derivatives can also be used. The arrangement of tamavidin is described in Japanese Patent No. 4257119, JP 2008-200038, and WO 2009/028625.
（５）投与方法及び製剤形態など
　本発明においては、腫瘍の診断又は治療を必要とする患者に、前記第一の標的化分子を投与し、前記第一の標的化分子が腫瘍部位に特異的に結合し、かつ当該腫瘍部位に安定して存在する間であって、前記患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間が経過した後に、前記第二の標的化分子を前記患者に投与し、前記放射性同位元素を前記腫瘍部位に局在化させる。 (5) Administration method, formulation form, etc. In the present invention, the first targeting molecule is administered to a patient in need of tumor diagnosis or treatment, and the first targeting molecule is specific to the tumor site. And the first targeting molecule adsorbed non-specifically to the non-tumor site of the patient is substantially excreted from the blood of the patient. After sufficient time has passed, the second targeting molecule is administered to the patient, and the radioisotope is localized at the tumor site.
　患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間は特に限定されないが、一般的には、前記第一の標的化分子の投与後６時間以上３０日以内であり、好ましくは６時間から２０日以内であり、より好ましくは６時間以上１６日以内であり、例えば、６時間から１４４時間、１２時間から９６時間、又は２４時間から９６時間である。また、前記第一の標的化分子の投与回数は特に限定されず、１回だけ投与してもよいし、２回以上の任意の回数（例えば、２～１０回など）で投与してもよい。十分な量の第一の標的化分子を腫瘍に集積させるためには、第一の標的化分子は２回以上の複数回投与することが好ましい。 The time sufficient for the first targeting molecule nonspecifically adsorbed to the non-tumor site of the patient to be substantially excreted from the patient's blood is not particularly limited, but generally the first targeting molecule is not limited. 6 hours to 30 days after administration of one targeting molecule, preferably 6 hours to 20 days, more preferably 6 hours to 16 days, for example, 6 hours to 144 hours, 12 hours From 96 hours, or from 24 hours to 96 hours. Further, the number of administrations of the first targeting molecule is not particularly limited, and it may be administered only once, or may be administered at any number of 2 times or more (for example, 2 to 10 times). . In order to accumulate a sufficient amount of the first targeting molecule in the tumor, the first targeting molecule is preferably administered two or more times.
　本発明で用いる、腫瘍細胞に特異的に結合する抗体断片と、一対の親和性物質のうちの一方とを連結した第一の標的化分子、及び放射性同位元素と、前記一対の親和性物質のうちの他方とを連結した第二の標的化分子は、それぞれ薬学的に許容しうる担体とともに、混合、溶解、乳化などすることによって、腫瘍の診断剤又は治療剤として用いてもよい。例えば、上記の第一の標的化分子及び第二の標的化分子は、薬学的に許容しうる溶媒、賦形剤、結合剤、安定化剤、分散剤等とともに、注射用溶液、懸濁液、乳剤等の剤形に製剤化することができる。注射用の処方においては、第一の標的化分子及び第二の標的化分子は、水性溶液、好ましくはハンクス溶液、リンゲル溶液、または生理的食塩緩衝液等の生理学的に適合性の緩衝液中に溶解することができる。さらに、組成物は、油性または水性のベヒクル中で、懸濁液、溶液、または乳濁液等の形状をとることができる。 A first targeting molecule in which an antibody fragment that specifically binds to tumor cells, one of a pair of affinity substances, and a radioisotope used in the present invention, and the pair of affinity substances The second targeting molecule linked to the other of them may be used as a diagnostic or therapeutic agent for tumors by mixing, dissolving, emulsifying, etc. together with a pharmaceutically acceptable carrier. For example, the first targeting molecule and the second targeting molecule can be used together with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc., injectable solutions, suspensions And can be formulated into a dosage form such as an emulsion. In an injectable formulation, the first targeting molecule and the second targeting molecule are in an aqueous solution, preferably a physiologically compatible buffer such as Hanks's solution, Ringer's solution, or physiological saline buffer. Can be dissolved. In addition, the composition can take the form of a suspension, solution, emulsion, or the like in an oily or aqueous vehicle.
　第一の標的化分子及び第二の標的化分子の投与経路は特に限定されないが、通常は非経口投与であり、例えば注射剤（皮下注、静注、筋注、腹腔内注など）、経皮、経粘膜などで投与することができる。 The route of administration of the first targeting molecule and the second targeting molecule is not particularly limited, but is usually parenteral administration, such as injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.) It can be administered through the skin or transmucosal membrane.
　投与量および投与回数は、患者の年齢、体重、診断の目的などによって異なる。一般には、第一の標的化分子及び第二の標的化分子の投与量としては、有効成分の投与量として１回あたり体重１ｋｇあたり、約０．１ｍｇから１０００ｍｇの範囲、好ましくは約０．１ｍｇから１００ｍｇの範囲となるように投与することができる。 The dose and number of doses vary depending on the patient's age, weight, and purpose of diagnosis. In general, the dosage of the first targeting molecule and the second targeting molecule is in the range of about 0.1 mg to 1000 mg, preferably about 0.1 mg per kg body weight per dose as the active ingredient dosage. To 100 mg can be administered.
　以下の実施例により本発明をさらに具体的に説明するが、本発明は実施例によって限定されるものではない。 The following examples further illustrate the present invention, but the present invention is not limited to the examples.
実施例１：改変モノクローナル抗体の作成
（１）ハイブリドーマ細胞よりのtotal RNAの調製]
　モノクローナル抗体B5209B (IgG2b)産生ハイブリドーマ細胞として、特開２００８－２９０９９６号公報に記載れているモノクローナル抗体Ｂ５２０９Ｂを産生するハイブリドーマを用いた。このモノクローナル抗体Ｂ５２０９Ｂを産生するハイブリドーマは、受託番号ＦＥＲＭ　Ｐ－２１２３８として、２００７年（平成１９年）３月２日付けで独立行政法人産業技術総合研究所　特許生物寄託センター（日本国茨城県つくば市東一丁目１番地１　中央第６（郵便番号３０５－８５６６））に寄託されており、さらに受託番号ＦＥＲＭ　ＢＰ－１０９２１として、２００７年（平成１９年）１０月１６日付けで国際寄託に移管された。 Example 1: Preparation of modified monoclonal antibody (1) Preparation of total RNA from hybridoma cells]
As a hybridoma cell producing monoclonal antibody B5209B (IgG2b), a hybridoma producing monoclonal antibody B5209B described in JP-A-2008-290996 was used. The hybridoma producing this monoclonal antibody B5209B has the accession number FERM P-21238 on March 2, 2007 (National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, East of Tsukuba City, Ibaraki Prefecture, Japan). 1-chome 1-address 1 Central No. 6 (postal code 305-8666), and was transferred to the international deposit on October 16, 2007 as deposit number FERM BP-10921 .
　上記のモノクローナル抗体B5209B (IgG2b)産生ハイブリドーマ細胞1 x 107個をphosphate -buffered saline (PBS)で一回洗浄後、細胞沈殿にTrizol液（Invitrogen社製）1 mlを加えて可溶化した。抽出液を20 G注射針を２回通してDNAをせん断した後、Trizol液付属の説明書に従ってクロロホルム抽出、イソプロパノール沈澱、80%エタノール洗浄によりtotal RNAを精製し、ジエチルピロカーボネート含有滅菌蒸留水に溶解した。得られたtotal RNAは、アガロースゲル電気泳動により、分解していないことを確認した。 The above monoclonal antibody B5209B (IgG2b) -producing 1 × 10 7 hybridoma cells were washed once with phosphate-buffered saline (PBS), and solubilized by adding 1 ml of Trizol solution (Invitrogen) to the cell precipitate. After the DNA was sheared by passing the extract through a 20 G injection needle twice, total RNA was purified by chloroform extraction, isopropanol precipitation, and 80% ethanol washing according to the instructions attached to the Trizol solution. Dissolved. It was confirmed that the obtained total RNA was not degraded by agarose gel electrophoresis.
（２）IgG heavy chain V region (VH) cDNAの合成とクローニング
　B5209B total RNA 5μgを鋳型として、3'-primerとしてマウスIgG2b heavy chain C領域5'端のcDNA配列に基づくprimer (5'-ccaagcttaggggccagtggatagactg-3')(配列番号５)を用い、SuperScript cDNA合成キット（Invitrogen社製）を使用してキットの説明に従い、1st strand cDNAを合成した。得られた1st strand cDNAにNovagen社Mouse Ig-Primer SetのMuIgVH5'-A primerを加えて、Expand High Fidelity PCR System (Roche Diagnostics社製)を用いて２本鎖cDNAを増幅した。得られた２本鎖cDNAをTAクローニング法によりpGEM-T vector (Promega社製)にサブクローニングし、大腸菌DH5αに導入してプラスミド含有クローンを得た。6クローンに関してプラスミドDNAをQiagen Plasmid Midi Kit (Qiagen社製)で精製し、常法に従って、DNA塩基配列を決定した。抗体の重鎖可変領域（ＶＨ）のアミノ酸配列は、配列番号２に記載のアミノ酸配列のうちの１番目から１２２番目までのアミノ酸配列（配列番号３のアミノ酸配列）であることが判明した。 (2) Synthesis and cloning of IgG heavy chain V region (VH) cDNA Using 5μg of B5209B total RNA as a template, 3′-primer based on the cDNA sequence of mouse IgG2b heavy chain C region 5 ′ end (5′-ccaagcttaggggccagtggatagactg- 3 ′) (SEQ ID NO: 5) was used to synthesize 1st strand cDNA according to the description of the kit using a SuperScript cDNA synthesis kit (manufactured by Invitrogen). MuIgVH5′-A primer of Novagen Mouse Ig-Primer Set was added to the obtained 1st strand cDNA, and double-stranded cDNA was amplified using Expand High Fidelity PCR System (Roche Diagnostics). The obtained double-stranded cDNA was subcloned into pGEM-T vector (Promega) by the TA cloning method and introduced into E. coli DH5α to obtain a plasmid-containing clone. Plasmid DNA of 6 clones was purified with Qiagen Plasmid Midi Kit (manufactured by Qiagen), and the DNA base sequence was determined according to a conventional method. The amino acid sequence of the heavy chain variable region (VH) of the antibody was found to be the amino acid sequence from the 1st to the 122nd amino acid sequence (amino acid sequence of SEQ ID NO: 3) of the amino acid sequence shown in SEQ ID NO: 2.
（３）抗ROBO1モノクローナル抗体B5209BのIgG light chainN末端アミノ酸配列の決定
　モノクローナル抗体B5209B (IgG2b)を含むハイブリドーマ無血清培養上清からProtein Gカラム（GE 社製）を用いて、付属の説明書に従い抗体の精製を行った。
　精製したモノクローナル抗体B5209BをSDS-PAGEを用いて電気泳動を行った。電気泳動ゲルはPVDF膜に転写した後、クマシー染色を行った。染色されたIgG Light chainのバンドを切り出し、エドマン分解法によりN末端アミノ酸配列（DIQMT）を決定した。 (3) Determination of IgG light chain N-terminal amino acid sequence of anti-ROBO1 monoclonal antibody B5209B Antibody from the hybridoma serum-free culture supernatant containing monoclonal antibody B5209B (IgG2b) using Protein G column (GE) according to the attached instructions Was purified.
The purified monoclonal antibody B5209B was electrophoresed using SDS-PAGE. The electrophoresis gel was transferred to a PVDF membrane and then stained with Coomassie. The stained IgG Light chain band was cut out and the N-terminal amino acid sequence (DIQMT) was determined by Edman degradation.
（４）B5209B mouse-scFv-SA の発現ベクターの構築（図１）
　図１に記載の構造を有するB5209B mouse-scFv-SA の発現ベクターを構築した。当該発現ベクター中のB5209B mouse-scFv-SA の塩基配列を配列番号１に記載し、アミノ酸配列を配列番号２に記載する。抗体の重鎖可変領域（ＶＨ）のアミノ酸配列は、配列番号２に記載のアミノ酸配列のうちの１番目から１２２番目までのアミノ酸配列（配列番号３のアミノ酸配列）に対応し、抗体の軽鎖可変領域（ＶＬ）のアミノ酸配列は、配列番号２に記載のアミノ酸配列のうちの１４２番目から２４８番目までのアミノ酸配列（配列番号４のアミノ酸配列）に対応する。 (4) Construction of B5209B mouse-scFv-SA expression vector (Figure 1)
An expression vector for B5209B mouse-scFv-SA having the structure shown in FIG. 1 was constructed. The base sequence of B5209B mouse-scFv-SA in the expression vector is described in SEQ ID NO: 1, and the amino acid sequence is described in SEQ ID NO: 2. The amino acid sequence of the heavy chain variable region (VH) of the antibody corresponds to the amino acid sequence from the 1st to the 122nd amino acid sequence (amino acid sequence of SEQ ID NO: 3) of the amino acid sequence shown in SEQ ID NO: 2, and the light chain of the antibody The amino acid sequence of the variable region (VL) corresponds to the amino acid sequence from the 142nd to the 248th amino acid sequence (amino acid sequence of SEQ ID NO: 4) of the amino acid sequence described in SEQ ID NO: 2.
（５）B5209B scFv-Streptavidin　培養法
　大腸菌BL21(DE3)を形質転換し、アンピシリン50μg/mL含有LBプレート培地にて28℃条件下で20時間程度培養した。プレートから単一コロニーを釣菌し、アンピシリン50 μg/mL含有LB試験管培地(3 mL)に植菌後、28℃にて18時間程度振とう培養(140 rpm程度)を行った。続いて、アンピシリン50μg/mL含有2×YT培地 (1L)に前培養液の全量を植え継ぎ、28℃にて振とう培養(125 rpm)した。OD600 = 0.8の時点で終濃度 0.5 mMのIPTGを添加することで発現誘導を行い、引き続き一晩培養した。 (5) B5209B scFv-Streptavidin culture method E. coli BL21 (DE3) was transformed and cultured in LB plate medium containing ampicillin 50 µg / mL for about 20 hours at 28 ° C. A single colony was picked from the plate, inoculated into LB test tube medium (3 mL) containing ampicillin 50 μg / mL, and then cultured with shaking (about 140 rpm) at 28 ° C. for about 18 hours. Subsequently, the entire amount of the preculture was transferred to 2 × YT medium (1 L) containing 50 μg / mL of ampicillin, and cultured with shaking (125 rpm) at 28 ° C. When OD 600 = 0.8, expression was induced by adding IPTG at a final concentration of 0.5 mM, followed by overnight culture.
（６）B5209B scFv-Streptavidin　調製法
　菌体内可溶性画分から目的蛋白質を回収し、Ni2+アフィニティーカラムHisTrap HP (GE healthcare)によって粗製性を行った。このとき、50 mM Tris-HCl, 200 mM NaCl, pH 8.0 緩衝液を移動相に用い、50 mM Tris-HCl, 200 mM NaCl, 500 mM イミダゾール, pH 8.0緩衝液を用いて段階的に溶出させた。目的蛋白質溶出画分を回収し、50 mM Tris-HCl, 200 mM NaCl, pH 8.0 緩衝液にて透析後、サイズ排除クロマトグラフィーにより最終精製を行った。使用したカラムはHiLoad TM 26/60 Superdex 200 (GE healthcare)、移動相には50 mM Tris-HCl, 200 mM NaCl, pH 8.0 緩衝液を用いた。最終精製物について、SDS-PAGEにより確認した。サイズ排除クロマトグラフィーによる最終精製とSDS-PAGEの結果を図２に示す。 (6) Preparation method of B5209B scFv-Streptavidin The target protein was recovered from the intracellular soluble fraction and subjected to crudeness using a Ni 2+ affinity column HisTrap HP (GE healthcare). At this time, 50 mM Tris-HCl, 200 mM NaCl, pH 8.0 buffer was used as the mobile phase, and eluted stepwise using 50 mM Tris-HCl, 200 mM NaCl, 500 mM imidazole, pH 8.0 buffer. . The target protein elution fraction was collected, dialyzed against 50 mM Tris-HCl, 200 mM NaCl, pH 8.0 buffer, and finally purified by size exclusion chromatography. The column used was HiLoad ™ 26/60 Superdex 200 (GE healthcare) and the mobile phase was 50 mM Tris-HCl, 200 mM NaCl, pH 8.0 buffer. The final purified product was confirmed by SDS-PAGE. The results of final purification by size exclusion chromatography and SDS-PAGE are shown in FIG.
実施例２：B5209B scFv-Streptavidin　活性評価
（１）ROBO1との結合活性
　等温滴定型熱量測定(ITC)により、B5209B scFv-StreptavidinとROBO1との相互作用に関する熱力学的解析を行った。図３には、溶媒にPBSを用い、25℃条件下にてB5209B scFv-Streptavidin (3.7μM)に対し、ROBO1(34.7μM)を一定量ずつ滴下した時の測定結果を示す。ここから算出された解離定数は5.6×10-8 (1/M)、エンタルピー変化量(ΔH)は-41.4 kJ/mol、またエントロピー変化量(ΔS)は0.52 J/mol・Kであった。scFvと比較すると、ΔHは顕著な変化は認められなかった。一方、ΔSは1/40程度低下しており、親和性ではおよそ一桁程度の低下が認められた。さらに、結合比では、B5209B scFv-Streptavidin 4量体に対しROBO1が2分子結合することが示唆され、4量体のうち隣接した2つの抗原結合部位は立体障害のため片方しかROBO1を認識できない可能性があると考えられる。 Example 2: Evaluation of B5209B scFv-Streptavidin activity (1) Binding activity with ROBO1 Thermodynamic analysis on the interaction between B5209B scFv-Streptavidin and ROBO1 was performed by isothermal titration calorimetry (ITC). FIG. 3 shows the measurement results when ROBO1 (34.7 μM) was added dropwise to B5209B scFv-Streptavidin (3.7 μM) under a condition of 25 ° C. using PBS as a solvent. The dissociation constant calculated from this was 5.6 × 10 −8 (1 / M), the enthalpy change (ΔH) was −41.4 kJ / mol, and the entropy change (ΔS) was 0.52 J / mol · K. Compared to scFv, ΔH did not change significantly. On the other hand, ΔS decreased by about 1/40, and the affinity decreased by about an order of magnitude. Furthermore, the binding ratio suggests that two molecules of ROBO1 bind to the B5209B scFv-Streptavidin tetramer, and two adjacent antigen-binding sites in the tetramer may be able to recognize only one ROBO1 due to steric hindrance. It is thought that there is sex.
（２）Biotinとの結合活性
　等温滴定型熱量測定(ITC)により、B5209B scFv-StreptavidinのBiotinに対する結合活性評価を行った。図４には、溶媒にPBSを用い、25℃条件下にてB5209B scFv-Streptavidin (9μM)に対し、Biotin (90μM)を一定量ずつ滴下した時の測定結果を示す。
　ここから算出された結合定数は6.94×10-9 (1/M)、エンタルピー変化量(ΔH)は-122.2 kJ/mol、また、エントロピー変化量(ΔS)は0.25 kJ/mol・Kであった。 (2) Binding activity to Biotin The binding activity of B5209B scFv-Streptavidin to Biotin was evaluated by isothermal titration calorimetry (ITC). FIG. 4 shows the measurement results when Biotin (90 μM) was added dropwise to B5209B scFv-Streptavidin (9 μM) under a condition of 25 ° C. using PBS as a solvent.
The binding constant calculated from this was 6.94 × 10 -9 (1 / M), the enthalpy change (ΔH) was -122.2 kJ / mol, and the entropy change (ΔS) was 0.25 kJ / mol · K. .
（３）B5209B scFv-Streptavidin　熱安定性評価
　示差走査型熱量測定(DSC)により、B5209B scFv-Streptavidinの熱安定性を評価した。そのときの測定結果を図５に示す。溶媒には、PBSを用いた。B5209B scFvの熱安定性は50℃付近であることから、B5209B scFv-StreptavidinのscFvドメインの変性温度はTm 51.4℃、Streptavidinドメイン4量体解離温度はTm 66.5℃、1分子の完全変性温度はTm 108℃であると推察される。 (3) Thermal stability evaluation of B5209B scFv-Streptavidin The thermal stability of B5209B scFv-Streptavidin was evaluated by differential scanning calorimetry (DSC). The measurement result at that time is shown in FIG. PBS was used as the solvent. Since the thermal stability of B5209B scFv is around 50 ° C, the denaturation temperature of B5209B scFv-Streptavidin's scFv domain is Tm 51.4 ° C, the streptavidin domain tetramer dissociation temperature is Tm 66.5 ° C, and the complete denaturation temperature of one molecule is Tm. Presumed to be 108 ° C.
実施例３：scFv-Streptavidinの血中濃度の測定
（１）マウス血清サンプルの調製
　HepG2細胞を移植した担癌ヌードマウスにscFv-Streptavidinを400μg投与した。15、30分後、1、2、4、8、24時間後に尾部から10μL採血を行い、3.2% クエン酸ナトリウム(wako)、1.6%ブロックエース(雪印乳業)をPBSに溶解したサンプルバッファーと混和して、血清サンプルとした。 Example 3 Measurement of blood concentration of scFv-Streptavidin (1) Preparation of mouse serum sample 400 μg of scFv-Streptavidin was administered to cancer-bearing nude mice transplanted with HepG2 cells. After 15, 30 minutes, 1, 2, 4, 8 and 24 hours later, 10 μL of blood is collected from the tail and mixed with a sample buffer in which 3.2% sodium citrate (wako) and 1.6% Block Ace (Snow Brand Milk Products) are dissolved in PBS. A serum sample was obtained.
（２）ELISAによるscFv-Streptavidin血中濃度の測定
　scFv-Streptavidin血中濃度の測定はReacti-Bind TM Biotin Coated PolystyreneStrip Plates (pierce)を用いて行った。以下にプロトコルを示す。Reacti-Bind TM Biotin Coated
Polystyrene Strip Platesへ、1.6%ブロックエースをPBSに溶解したブロッキングバッファーを添加し、室温で１時間静置してブロッキングを行った。0.05% Tween20をPBSに溶解した洗浄バッファーで洗浄後、ブロッキングバッファーで希釈した血清サンプルを添加し、室温で１時間静置してscFv-Streptavidinをプレートへ吸着させた。洗浄バッファーで洗浄後、ブロッキングバッファーで希釈したHRP標識抗ストレプトアビジン抗体(abcam)を添加し、室温で１時間静置して抗体反応を行った。洗浄バッファーで洗浄後、TMB発色試薬(scytek)を添加し、室温で30分間反応させた。反応後、TMB発色停止液を添加し、450nm吸光度を測定した。血清サンプル中のscFv-Streptavidin濃度は、精製Streptavidin (pierce)の希釈系列より検量線を作製し、算出した。結果を図１３に示す。横軸はscFvSAを投与してからの時間、縦軸は投与してから15分後のscFvSA濃度を100％としたときそれぞれの時間における濃度を%で表す。 (2) Measurement of scFv-Streptavidin blood concentration by ELISA Measurement of scFv-Streptavidin blood concentration was performed using Reacti-Bind ™ Biotin Coated Polystyrene Strip Plates (pierce). The protocol is shown below. Reacti-Bind TM Biotin Coated
A blocking buffer prepared by dissolving 1.6% block ace in PBS was added to Polystyrene Strip Plates and allowed to stand at room temperature for 1 hour for blocking. After washing with a washing buffer in which 0.05% Tween 20 was dissolved in PBS, a serum sample diluted with a blocking buffer was added, and the mixture was allowed to stand at room temperature for 1 hour to adsorb scFv-Streptavidin to the plate. After washing with a washing buffer, an HRP-labeled anti-streptavidin antibody (abcam) diluted with a blocking buffer was added, and the mixture was allowed to stand at room temperature for 1 hour for antibody reaction. After washing with the washing buffer, TMB coloring reagent (scytek) was added and reacted at room temperature for 30 minutes. After the reaction, TMB color development stop solution was added, and the absorbance at 450 nm was measured. The scFv-Streptavidin concentration in the serum sample was calculated by preparing a calibration curve from a dilution series of purified Streptavidin (pierce). The results are shown in FIG. The horizontal axis represents the time after scFvSA administration, and the vertical axis represents the concentration at each time in% when the scFvSA concentration 15 minutes after administration is taken as 100%.
　また、scFv-Streptavidin血中濃度の測定結果の対数近似曲線を図１４に示す。
Y = -18.24ln(x) + 75.107
R2 = 0.9832
図１４に示す近似曲線より48, 72, 168時間後の血中濃度を推定すると
2 days (48 h): Y = -18.24ln(48) + 75.107 = 4.50 (%)
3 days (72 h): Y = -18.24ln(48) + 75.107 = -2.90 (%)
7 days (168 h): Y = -18.24ln(48) + 75.107 = -18.35 (%)
となる。 In addition, a logarithmic approximation curve of the measurement result of scFv-Streptavidin blood concentration is shown in FIG.
Y = -18.24ln (x) + 75.107
R2 = 0.9832
From the approximate curve shown in FIG. 14, the blood concentration after 48, 72, and 168 hours is estimated.
2 days (48 h): Y = -18.24ln (48) + 75.107 = 4.50 (%)
3 days (72 h): Y = -18.24ln (48) + 75.107 = -2.90 (%)
7 days (168 h): Y = -18.24ln (48) + 75.107 = -18.35 (%)
It becomes.
実施例４：マイクロPET
（１）材料と方法
改変モノクローナル抗体：
　実施例１で作製した抗Robo1 IgG B5209 mAb (150kDa)からscFv-StreptAvdin (scFv-SA, SA四量体を含めて170kDa)を使用した(図６)。なお、scFvは28kDaである。なお、いずれのモノクローナル抗体（mAb）についても、抗体結合活性が低下していないことを事前に確認し、マイクロPET実験の際には、マウス一匹あたり400～1000μgを使用した。 Example 4: Micro PET
(1) Material and method modified monoclonal antibody:
From the anti-Robo1 IgG B5209 mAb (150 kDa) prepared in Example 1, scFv-StreptAvdin (scFv-SA, 170 kDa including SA tetramer) was used (FIG. 6). ScFv is 28 kDa. For any monoclonal antibody (mAb), it was confirmed in advance that the antibody binding activity was not lowered, and 400 to 1000 μg per mouse was used in the microPET experiment.
使用動物と前処置：
　HepG2細胞107個を6週齢のBALB/cAjcl-nu/nu雄マウスに移植し、マイクロPET実験時に12週齢、体重約25ｇとなるようにxenograft nude mouse modelを作成した。腫瘤部のサイズは8～10mmである。すべてのマウスをポジトロン核種投与前の6時間は絶食とし、飲水のみとした。なお、プレターゲッティング実験の場合には、内因性Biotinの低減を目的として、マイクロPET実験の約一週間前から低ビオチン食を給餌した。 Animals used and pretreatment:
10 7 HepG2 cells were transplanted into 6-week-old BALB / cAjcl-nu / nu male mice, and a xenograft nude mouse model was prepared so that the body weight was about 25 g at 12 weeks of age in the microPET experiment. The size of the mass is 8-10 mm. All mice were fasted for 6 hours before positron radionuclide administration and only drinking water. In the case of the pretargeting experiment, a low biotin diet was fed about one week before the microPET experiment for the purpose of reducing endogenous Biotin.
放射性医薬品の合成：
　ポジトロン核種64Cu(半減期12.7時間)を、サイクロトロンにて64Ni(p,n) 64Cu核反応を生じさせる方法により製造した。次に、製造した比放射能の高い64Cuを、双機能性キレート剤1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA)を介して、事前に作成したプレターゲッティング用cold DOTA-Biotinの標識に使用した。 Radiopharmaceutical synthesis:
The positron nuclide 64 Cu (half-life 12.7 hours) was produced by a method that causes a 64 Ni (p, n) 64 Cu nuclear reaction in a cyclotron. Next, 64 Cu with high specific activity was prepared in advance via the bifunctional chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Used for labeling cold DOTA-Biotin for lettergetting.
使用機器およびデータ収集：
　positron emissionデータ収集には、Siemens社製小動物用スキャナーマイクロPET Focus 120を用いた。ヌードマウス尾静脈に留置したラインから64Cu標識放射性医薬品（プレターゲッティングの場合、64Cu-DOTA-Biotin）を投与し、Emissionデータを収集した。 Equipment used and data collection:
For collecting positron emission data, a micro animal scanner Micro PET Focus 120 manufactured by Siemens was used. 64 Cu-labeled radiopharmaceutical ( 64 Cu-DOTA-Biotin in the case of pretargeting) was administered from a line placed in the nude mouse tail vein, and emission data was collected.
Study 1: 64Cu-DOTA-Biotin-SA-scFv複合体マイクロPET Biodistribution試験（参考例）
　scFv-SAの腫瘍細胞表面抗原への結合性を確認するために、scFv-SAの直接標識放射性医薬品を64Cu-DOTA-Biotin溶液と生体外で直接混和することによって作成し、xenograft nude mouseに投与した。64Cu-DOTA-Biotin-SA-scFv（複合体）の投与開始から最大で約4日後まで、マイクロPETを用いて64Cu標識複合体のヌードマウス全身への分布画像を作成した。 Study 1: 64 Cu-DOTA-Biotin-SA-scFv complex micro PET Biodistribution test (reference example)
To confirm the binding of scFv-SA to tumor cell surface antigens, a scFv-SA directly labeled radiopharmaceutical was prepared by mixing in vitro with 64 Cu-DOTA-Biotin solution in vitro and transferred to xenograft nude mice. Administered. From the start of administration of 64 Cu-DOTA-Biotin-SA-scFv (complex) to a maximum of about 4 days later, a distribution image of 64 Cu-labeled complex throughout the body of nude mice was created using microPET.
Study 2: 64Cu-DOTA-Biotin マイクロPET Biodistribution試験（参考例）
　プレターゲッティングに用いられるBiotin-Avdinの特異的結合は極めて強固であるが、DOTA-BiotinがscFv-SAとの結合に寄与しない場合も一定の割合で想定される。64Cu-DOTA-Biotin単独のbiodistributionを知るため、scFv-SAを投与せず64Cu-DOTA-BiotinのみHepG2腫瘍マウスモデルに投与し、マイクロPET イメージング を実施した。 Study 2: 64 Cu-DOTA-Biotin Micro PET Biodistribution Test (Reference Example)
The specific binding of Biotin-Avdin used for pregetting is extremely strong, but it is also assumed at a certain rate that DOTA-Biotin does not contribute to the binding with scFv-SA. In order to know the biodistribution of 64 Cu-DOTA-Biotin alone, scFv-SA was not administered and only 64 Cu-DOTA-Biotin was administered to the HepG2 tumor mouse model, and microPET imaging was performed.
Study 3: 64Cu-DOTA-Biotin & scFv-SA プレターゲッティング 試験（clearing agentを使用）（参考例）
　clearing agentはプレターゲッティング実験において癌特異的抗原と結合していない血中の余剰抗体を除去する目的に用いられる。血液中からのクリアリングは肝細胞に潤沢なアシアロ糖蛋白レセプターを介する。余剰のscFv-SAとの結合はclearing agentにも付加されたbiotinによる。つまり、Biotin-SA結合は、余剰抗体のclearing にも、pretargetされた癌細胞表面抗原の二次的な標識（腫瘍のイメージング）にも用いられていることになる。64Cu投与予定時刻の約一日前にscFvを、引き続いて64Cu投与の一定時間前には余剰の血中scFvを除去する目的で、Clearing Agentを投与した。具体的には、約3分前から12時間前までの複数の投与パターンを検討している。Clearing Agentとして99mTc標識前のアシアロシンチ原末（日本メジフィジックス社製ガラクトシル人血清アルブミン）を入手し、Biotin化した上でマウス一匹につき約100-1000μgを使用した。 Study 3: 64 Cu-DOTA-Biotin & scFv-SA Pre-lettering test (using a clearing agent) (reference example)
The clearing agent is used for the purpose of removing excess antibodies in the blood that are not bound to the cancer-specific antigen in pretargeting experiments. Clearing from the blood is mediated by asialoglycoprotein receptors abundant in hepatocytes. Binding to surplus scFv-SA is due to biotin added to the clearing agent. In other words, Biotin-SA binding is used both for clearing excess antibodies and for secondary labeling of pretargeted cancer cell surface antigens (tumor imaging). A clearing agent was administered for the purpose of removing scFv about a day before the scheduled time of 64 Cu administration and subsequently removing excess blood scFv at a certain time before 64 Cu administration. Specifically, we are studying multiple dosing patterns from about 3 minutes to 12 hours ago. Asialosinchi bulk powder (galactosyl human serum albumin manufactured by Japan Mediphysix) before 99m Tc labeling was obtained as a Clearing Agent, biotinylated, and about 100-1000 μg per mouse was used.
Study 4: 64Cu-DOTA-Biotin & scFv-SA プレターゲッティング 試験（clearing agentを使用しない）（本発明）
　生体に投与されたclearing agnetが仮に分解され、遊離のBiotinを生じたと仮定すると癌細胞表面にpretargetされた改変抗体のSA結合部位を占拠してしまう。これは腫瘍イメージングには有害な作用である。よって、Clearing agent(CA)を使用せずにプレターゲッティングによるマイクロPET イメージングを行った。別途、実験にてscFv-SAの血液中半減期が約8時間であることが判明しており、これに基づけばCAを使用せずにscFv-SAの十分な血中クリアランスを得るためには最低2日の投与間隔が必要となる。よって、64Cu-DOTA-Biotin投与の約3日前にscFv-SAのみを約1000μg投与し、マイクロPET イメージング を実施した。なお、この例では、腎集積を減らすための利尿負荷をかけることはしていない。 Study 4: 64 Cu-DOTA-Biotin & scFv-SA Pre-lettering test (no clearing agent used) (Invention)
Assuming that the clearing agnet administered to the living body is decomposed to produce free Biotin, the SA binding site of the modified antibody pretargeted on the cancer cell surface is occupied. This is a detrimental effect on tumor imaging. Therefore, micro-PET imaging by pretargeting was performed without using the Clearing agent (CA). Separately, it has been found in experiments that the blood half-life of scFv-SA is about 8 hours. Based on this, in order to obtain sufficient blood clearance of scFv-SA without using CA A minimum administration interval of 2 days is required. Therefore, about 1000 μg of scFv-SA alone was administered about 3 days before 64 Cu-DOTA-Biotin administration, and microPET imaging was performed. In this example, no diuretic load is applied to reduce renal accumulation.
画像再構成と画像データ解析（概略）：
　収集したPETデータは、上記のStudy1、Study2、Study3、およびStudy4以降ともに一連の処理後、最終段階でFiltered Back Projection法によって、3Dもしくは4D画像(dynamicデータ収集の場合)へと再構成した。画像表示、臓器や腫瘍の関心領域（ROI）設定は、マイクロPET用ソフトウェアであるAsiProなどを用いて行った。 Image reconstruction and image data analysis (outline):
The collected PET data was reconstructed into a 3D or 4D image (in the case of dynamic data collection) by the Filtered Back Projection method at the final stage after a series of processes for Study 1, Study 2, Study 3, and Study 4 and later. Image display and organ / tumor region of interest (ROI) settings were made using AsiPro, a microPET software.
（２）結果
Study 1: 64Cu-DOTA-Biotin-SA-scFv複合体マイクロPET Biodistribution試験（参考例）
　図７Ａでは、64Cu-DOTA-Biotin-SA-scFv complex投与後2時間にても64Cu-DOTA-Biotin-SA-scFv複合体の血中滞留性は高い。ほとんどが肝臓経由で排泄され、分子サイズの大きさのため腫瘍への移行性は低いと推察される(図７Ｂ、Ｃ)。複合体分子の腫瘍への移行は量的には少ないが、3日後まで緩徐に腫瘍部の集積は上昇し、正常軟部組織と比べた場合には腫瘍への特異的結合を確認することができた(図７Ｄ)。 (2) Results
Study 1: 64 Cu-DOTA-Biotin-SA-scFv complex micro PET Biodistribution test (reference example)
In Figure 7A, retention in blood of 64 64 also in Cu-DOTA-Biotin-SA- scFv complex 2 hours after administration-Cu-DOTA Biotin-SA- scFv complexes is higher. Most of them are excreted via the liver, and due to the large molecular size, the migration to the tumor is assumed to be low (FIGS. 7B and 7C). Although the amount of complex molecules transferred to the tumor is small, the accumulation of the tumor gradually increases until 3 days later, and specific binding to the tumor can be confirmed when compared with normal soft tissue. (FIG. 7D).
Study 2: 64Cu-DOTA-Biotin マイクロPET Biodistribution試験（参考例）
　64Cu-DOTA-Biotinの単独投与後30分では膀胱へすでに排泄されたRI、腎盂に貯留しているRI（両側腎臓中央部）、腎実質に集積しているRI、および肝実質に集積していると考えられるRIが描出されている(図８Ａ)。2時間後では腎尿路系から64Cu-DOTA-Biotinはほとんど排泄されているにもかかわらず、肝臓への集積が持続している(図８Ｂ)。一日後では、胆道系を介して腸管へ排泄されたと考えられるRI像に加えて、新たに腫瘍への集積が同定できる。肝臓への集積は依然として最も高い(図８Ｃ)。なお、肝への集積度は10～15%ID/gと算出される。 Study 2: 64 Cu-DOTA-Biotin Micro PET Biodistribution Test (Reference Example)
In 30 minutes after single administration of 64 Cu-DOTA-Biotin, RI already excreted in the bladder, RI accumulated in the renal pelvis (bilateral central kidney), RI accumulated in the renal parenchyma, and accumulated in the liver parenchyma The RI considered to be present is depicted (FIG. 8A). After 2 hours, 64 Cu-DOTA-Biotin is almost excreted from the renal urinary tract system, but accumulation in the liver continues (FIG. 8B). One day later, in addition to the RI image thought to have been excreted into the intestinal tract via the biliary system, a new accumulation in the tumor can be identified. Accumulation in the liver is still the highest (FIG. 8C). The degree of accumulation in the liver is calculated as 10-15% ID / g.
Study 3: 64Cu-DOTA-Biotin & scFv-SA プレターゲッティング試験（clearing agentsを使用）（参考例）
　図９は64Cu-DOTA-Biotinの投与36時間前にscFv-SAを400μg投与し、12時間前にclearing agentを1000μg投与したヌードマウスの例である。腫瘍へのRIの集積度は低く、むしろCAはこの例では集積度を下げており、CA使用の有用性は認められない。Clearing agentの投与方法については、64Cu-DOTA-Biotin投与までの間隔が一時間以内の場合から、12時間まで、さらにCAの投与量も増減させて複数の場合を検討したが、腫瘍部への集積が他の正常臓器と比較して良好となる結果を得ることはできなかった。 Study 3: 64 Cu-DOTA-Biotin & scFv-SA Pre-lettering test (using clearing agents) (reference example)
FIG. 9 shows an example of a nude mouse administered with 400 μg of scFv-SA 36 hours before administration of 64 Cu-DOTA-Biotin and 1000 μg of clearing agent 12 hours before administration. The accumulation of RI in the tumor is low, rather the CA has decreased in this example, and the usefulness of using CA is not observed. Regarding the administration method of the clearing agent, we examined multiple cases where the interval between administrations of 64 Cu-DOTA-Biotin was within one hour, to 12 hours, and the CA dose was increased or decreased. It was not possible to obtain a result in which the accumulation of was better than other normal organs.
Study 4: 64Cu-DOTA-Biotin & scFv-SA プレターゲッティング 試験（clearing agentを使用しない）（本発明）
　64Cu-DOTA-Biotin投与から一日後の画像では、概ね腫瘍、肝臓、腎臓の集積が同程度どなっているが、腎集積がやや優位である。時間の経過とともに腫瘍への集積は増強し、一方で肝臓、腎臓からの排泄が進む結果、腫瘍への集積が最終的には優位となっている(図１０)。結局、CAを用いるStudy3のすべての実験と比較しても、腫瘍への集積度に関してCAを使用しないStudy 4の結果を上回る例は確認できなかった。 Study 4: 64 Cu-DOTA-Biotin & scFv-SA Pre-lettering test (no clearing agent used) (Invention)
In the image one day after 64 Cu-DOTA-Biotin administration, the accumulation of tumor, liver, and kidney is almost the same, but renal accumulation is somewhat superior. Accumulation in the tumor increases with time, while excretion from the liver and kidney progresses. As a result, accumulation in the tumor is finally dominant (FIG. 10). Eventually, even when compared with all the studies of Study 3 using CA, we could not confirm any examples that exceeded the results of Study 4 that did not use CA in terms of tumor accumulation.
　scFv-SA投与から64Cu-DOTA-Biotin投与までの間隔を変化させて、腫瘍集積への影響を評価した(図１１)。その結果、投与間隔4日間、10日間、１６日間いずれも腫瘍への集積性は保たれており、比較的長期間にわたってscFv-SAが細胞表面から外れない・除去されない(あるいはinternalizationされない)ということが示され、scFv-SAと64Cu-DOTA-Biotinの投与間隔と投与方法を工夫することで、血中の余剰抗体の低減化とscFv-SAの腫瘍への高集積化を同時に達成できる可能性が示唆された。 The interval from scFv-SA administration to 64 Cu-DOTA-Biotin administration was changed to evaluate the effect on tumor accumulation (FIG. 11). As a result, the accumulation in the tumor is maintained for every 4 days, 10 days, and 16 days, and scFv-SA is not removed from the cell surface or removed (or not internalized) for a relatively long period of time. By devising the administration interval and administration method of scFv-SA and 64 Cu-DOTA-Biotin, it is possible to simultaneously reduce the excess antibody in the blood and increase the accumulation of scFv-SA in the tumor. Sex was suggested.
Study 5: 90Y-DOTA-Biotin ＆　scFv-SAプレターゲッテイング試験( clearing agentを使用しない)(本発明)
　HepG2担癌マウスに、プレターゲットのため、scFv-SA400μgを尾静脈より投与し、その４日後に90Yを標識したDOTA-Biotin  1.5mCiを投与し、腫瘍増殖抑制効果の有無を評価した。図１２に90Y-DOTA-Biotin投与後、30日間の腫瘍増殖曲線を示す。Ｙ軸は、投与日の腫瘍体積を１としたときの腫瘍体積比、X軸は投与後の時間（日）を示す。概ね、投与したマウスの腫瘍増殖曲線は、コントロールマウスの近似増殖曲線（太線で表示：scFv-SA 400μgのみ投与し、90Y-DOTA-Biotinを投与していない群）を下回り、腫瘍増殖がコントロール群に比し抑えられた。また、30日の時点で、投与マウスの健康状態は、比較的良好であった。このことから、クリアリングエージェントを用いないプレターゲット法で、重篤な副作用を出さずに、腫瘍増殖をある程度抑制出来ることが示唆された。 Study 5: 90 Y-DOTA-Biotin & scFv-SA pretargeting test (no clearing agent) (invention)
For pretargeting, scFv-SA 400 μg was administered to HepG2 tumor-bearing mice from the tail vein, and 90 Y-labeled DOTA-Biotin 1.5 mCi was administered 4 days later, and the presence or absence of a tumor growth inhibitory effect was evaluated. FIG. 12 shows a tumor growth curve for 30 days after 90 Y-DOTA-Biotin administration. The Y axis shows the tumor volume ratio when the tumor volume on the administration day is 1, and the X axis shows the time (day) after administration. In general, the tumor growth curve of the administered mice is lower than the approximate growth curve of the control mice (indicated by bold line: only 400 μg of scFv-SA and no 90 Y-DOTA-Biotin is administered), and tumor growth is controlled Controlled compared to the group. In addition, at 30 days, the health of the administered mice was relatively good. This suggests that the pretargeting method without a clearing agent can suppress tumor growth to some extent without causing serious side effects.
（３）考察
　固形腫瘍のイメージングあるいは治療を目的とする分子プローブとして、モノクローナル抗体(mAb)を用いる場合には、IgGの長い血中滞留性は正常組織への高い分布による腫瘍イメージングの際のコントラスト低下のみならず、RITに応用した場合には最も放射線感受性の高い造血器の被ばく障害という致命的な問題を生じる。したがって、改変mAbの導入は固形腫瘍を対象とするイメージングおよび治療用の分子プローブを開発する上で必須である。改変mAbの生体内分布など薬物としての基本的な動態は、正常臓器への悪影響を最小限に抑制し、かつ腫瘍部へのイメージング/治療の効果が最大限に発揮されるように最適化されていなくてはならない。本実施例では、肝細胞癌に高発現しているRobo1を標的とするmAb分子B5209 IgGを元に抗体の改変を進め、機能的最小fragmentであるscFvを得た。さらに、64Cuの比放射能が低かった問題を克服した上で、HepG2腫瘍ヌードマウスを対象にしてプレターゲッティング法の導入を行った。Goldenbergらの総説によれば、Biotin-SA系を用いたプレターゲッティング法の場合には、癌細胞表面抗原との結合していない血中の余剰抗体を低減するためにclearing agentの使用が標準となっている。しかしながら、本発明によれば、biotinをcomponentとして含むclearing agentの使用が、必ずしも64Cu-DOTA-Biotinを用いたマイクロPETイメージングに有益であるとは限らないことが示された。腫瘍細胞上のscFv-SAにあるビオチン結合部位をclearing agent に由来するビオチンが少量であれ占拠している可能性がある。 (3) Discussion When monoclonal antibodies (mAbs) are used as molecular probes for imaging or treatment of solid tumors, the long retention of IgG in the blood is due to the high distribution in normal tissues. In addition to the reduction, when applied to RIT, the most radiosensitive hematopoietic exposure damage is fatal. Therefore, the introduction of modified mAbs is essential in developing molecular probes for imaging and treatment of solid tumors. The basic kinetics of the modified mAb such as biodistribution are optimized to minimize adverse effects on normal organs and maximize the effects of imaging / treatment on the tumor site. It must be. In this example, the modification of the antibody was advanced based on the mAb molecule B5209 IgG targeting Robo1, which is highly expressed in hepatocellular carcinoma, to obtain scFv which is a functional minimum fragment. Furthermore, after overcoming the problem of low specific activity of 64 Cu, we introduced a pretargeting method in nude mice with HepG2 tumors. According to a review by Goldenberg et al., In the case of pretargeting using the Biotin-SA system, the use of a clearing agent is the standard to reduce excess antibodies in the blood that are not bound to cancer cell surface antigens. It has become. However, according to the present invention, it was shown that the use of a clearing agent containing biotin as a component is not necessarily beneficial for micro PET imaging using 64 Cu-DOTA-Biotin. The biotin-binding site in scFv-SA on tumor cells may occupy a small amount of biotin derived from the clearing agent.
　clearing agent を使用せずにプレターゲッティング法を実施する場合には、scFv-SAが自然にクリアランスされて十分に血中濃度が低下するまで、64Cu-DOTA-Biotinの投与を待たなくてはならないが、その一方で投与物質の数（すなわち投与回数）が減ることによるメリットがある。特に生体内でscFv-SAと64Cu-DOTA-Biotinの間にどのようなinteractionが生じているのか考えやすい。今後さらに抗体の改変を進めたり、プレターゲッティング systemの改善を図る上で、これは大きな利点となりうる。具体的にscFv-SAと64Cu-DOTA-Biotinの投与によって血中に生じるものは、少量の複合体と、極少量のフリーの64Cu-DOTA-Biotinであると思われるが、これらも図７及び図８で示されているように緩徐に腫瘍への集積するため、腫瘍イメージングの大きな妨げにはならなかったと思われる。 When pretargeting is performed without using a clearing agent, 64 Cu-DOTA-Biotin must be waited until scFv-SA is naturally cleared and the blood concentration is sufficiently reduced. On the other hand, there is a merit due to a decrease in the number of administered substances (that is, the number of administrations). In particular, it is easy to consider what kind of interaction occurs between scFv-SA and 64 Cu-DOTA-Biotin in vivo. This can be a great advantage for further antibody modifications and improvements to the pretargeting system. Specifically, what is produced in the blood by administration of scFv-SA and 64 Cu-DOTA-Biotin seems to be a small amount of complex and a very small amount of free 64 Cu-DOTA-Biotin. 7 and FIG. 8, as it slowly accumulates in the tumor, it does not appear to have been a major obstacle to tumor imaging.
　64Cuを用いたマイクロPET イメージングから90Yが100%放射するベータ線を活用してRITを目指した場合、最も重要なことは、放射線感受性の低い正常臓器（特に造血器）への集積を抑制して、すべての正常臓器を被ばく許容閾値以下に保つ一方、腫瘍部への集積を最大限にすることである。腫瘍への集積は特異的な抗原抗体結合のみに依存する必要はなく、RIT目的では64Cu-DOTA-Biotin単独で示した腫瘍集積のように何からのpassiveなメカニズムによる集積であってもよい。上記Study4のbiodistributionからは、whole IgGよりも造血器の被ばくが著しく低下したことが明らかである。さらFab抗体の直接標識では高いFabの腎集積が明らかであったが、プレターゲッティング法によって64Cu-DOTA-Biotinを使用する場合には利尿負荷によって腎集積をさらに減少させることが可能である。以上により、正常組織の被ばくを抑えながら、一定の腫瘍への内照射治療効果を期待することができる。 When aiming for RIT using beta-radiation that emits 100% of 90 Y from micro-PET imaging using 64 Cu, the most important thing is to suppress accumulation in normal organs (especially hematopoietic organs) with low radiosensitivity Thus, while keeping all normal organs below the exposure threshold, the accumulation in the tumor is maximized. Tumor accumulation need not depend solely on specific antigen-antibody binding, and for RIT purposes it can be from any passive mechanism, such as tumor accumulation shown with 64 Cu-DOTA-Biotin alone . From the Study4 biodistribution, it is clear that hematopoietic exposure was significantly lower than whole IgG. Furthermore, high Fab renal accumulation was evident by direct labeling of the Fab antibody, but when 64 Cu-DOTA-Biotin is used by the pretargeting method, renal accumulation can be further reduced by diuretic load. As described above, it is possible to expect an internal irradiation treatment effect on a certain tumor while suppressing exposure of a normal tissue.
　実際のＲＩＴにおいては、正常臓器への被曝を最小限に抑制し、腫瘍増殖抑制・縮小効果を得ることが重要である。本ＲＩＴ実施例においては、抗ROBO1改変抗体による、クリアリングエージェントを用いないプレターゲット法によるRITを施行し、重篤な副作用を出さずに、腫瘍増殖をある程度抑制出来ることが示唆された。プレターゲットによるRITにおいて高い抗腫瘍効果の達成には、当然ながら腫瘍への核種の高集積化が必要である。その方法の一つとして、核種投与量の増量が挙げられるが、主として全身への放射線障害の観点から90Y-DOTA-Biotinの投与量の上限が決定されることとなる。核種の高集積化を達成する別の方法としては、生体への影響を与えない程度量の改変抗体を間隔をおいて数回投与して腫瘍への高集積化を達成させるブースト法が考えられる。このブースト法の必要条件として、改変抗体がある程度長い期間、腫瘍表面に集積し続ける必要がある。本実施例では投与間隔4日間、10日間、１６日間いずれも腫瘍への集積性は保たれており、比較的長期間にわたってscFv-SAが細胞表面から外れない・除去されないことが示され、このことからブースト投与によるscFv-SAの高集積化が達成される可能性があるということが示された。このブースト投与法とＲＩＴを組み合わせることで、さらに高い抗腫瘍効果が得られることが期待される。 In actual RIT, it is important to minimize exposure to normal organs and to obtain a tumor growth suppression / reduction effect. In this RIT example, it was suggested that RIT by a pretargeting method without using a clearing agent with an anti-ROBO1 modified antibody was performed, and tumor growth could be suppressed to some extent without causing serious side effects. In order to achieve a high anti-tumor effect in pre-targeted RIT, it is natural that high accumulation of nuclides in the tumor is necessary. One method is to increase the dose of the nuclide, and the upper limit of the dose of 90 Y-DOTA-Biotin is determined mainly from the viewpoint of radiation damage to the whole body. As another method for achieving high accumulation of nuclides, a boosting method in which high accumulation in the tumor is achieved by administering a modified antibody in an amount that does not affect the living body several times at intervals is conceivable. . A prerequisite for this boost method is that the modified antibody must continue to accumulate on the tumor surface for some length of time. In this example, it was shown that scFv-SA was not removed from or removed from the cell surface over a relatively long period of time, because the accumulation in the tumor was maintained for every 4 days, 10 days, and 16 days. From this, it was shown that scFv-SA can be highly integrated by boost administration. It is expected that a higher antitumor effect can be obtained by combining this boost administration method and RIT.

Claims (30)

  1. （ａ）腫瘍細胞に特異的に結合する抗体断片と、一対の親和性物質のうちの一方とを連結した第一の標的化分子、及び（ｂ）放射性同位元素と、前記一対の親和性物質のうちの他方とを連結した第二の標的化分子を含む、腫瘍の診断剤又は治療剤キットであって、腫瘍の診断又は治療を必要とする患者に、前記第一の標的化分子を投与し、前記第一の標的化分子が腫瘍部位に特異的に結合し、かつ当該腫瘍部位に安定して存在する間であって、前記患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間が経過した後に、前記第二の標的化分子を前記患者に投与し、前記放射性同位元素を前記腫瘍部位に局在化させることを特徴とする、上記の腫瘍の診断剤又は治療剤キット。 (A) a first targeting molecule in which an antibody fragment that specifically binds to a tumor cell and one of a pair of affinity substances are linked; and (b) a radioisotope and the pair of affinity substances. A diagnostic or therapeutic agent kit for a tumor comprising a second targeting molecule linked to the other of the two, wherein the first targeting molecule is administered to a patient in need of diagnosis or treatment of the tumor The first targeting molecule binds specifically to the tumor site and is stably present at the tumor site, and is non-specifically adsorbed to the non-tumor site of the patient. After a sufficient amount of time has elapsed for one targeting molecule to be substantially excreted from the patient's blood, the second targeting molecule is administered to the patient and the radioisotope is delivered to the tumor site. A diagnostic or therapeutic agent kit for a tumor as described above, which is localized.
  2. 一対の親和性物質のうちの一方が、ビオチン結合性タンパク質であり、一対の親和性物質のうちの他方がビオチン又はその誘導体である、請求項１に記載の腫瘍の診断剤又は治療剤キット。 The diagnostic or therapeutic agent kit for tumors according to claim 1, wherein one of the pair of affinity substances is a biotin-binding protein and the other of the pair of affinity substances is biotin or a derivative thereof.
  3. ビオチン結合性タンパク質が、アビジン、ストレプトアビジン、タマビジン、又はそれらの誘導体である、請求項２に記載の腫瘍の診断剤又は治療剤キット。 The diagnostic or therapeutic agent kit for tumors according to claim 2, wherein the biotin-binding protein is avidin, streptavidin, tamavidin, or a derivative thereof.
  4. 第二の標的化分子において、放射性同位元素と結合しうるキレート剤を用いて、放射性同位元素と前記一対の親和性物質のうちの他方とが連結している、請求項１から３の何れかに記載の腫瘍の診断剤又は治療剤キット。 The radioisotope and the other of the pair of affinity substances are linked to each other using a chelating agent capable of binding to the radioisotope in the second targeting molecule. A diagnostic or therapeutic agent kit for a tumor according to 1.
  5. 腫瘍細胞に特異的に結合する抗体断片が、抗Ｒｏｂｏ１抗体である、請求項１から４の何れかに記載の腫瘍の診断剤又は治療剤キット。 The diagnostic or therapeutic agent kit for a tumor according to any one of claims 1 to 4, wherein the antibody fragment that specifically binds to a tumor cell is an anti-Robo1 antibody.
  6. 第一の標的化分子が、腫瘍細胞に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）と、ストレプトアビジとを連結してなる４量体又は２量体の分子である、請求項１から５の何れかに記載の腫瘍の診断剤又は治療剤キット。 The first targeting molecule is a tetramer or dimer molecule obtained by linking a single-chain antibody fragment (scFv) that specifically binds to a tumor cell and streptavidin. To 5. The diagnostic or therapeutic agent kit for a tumor according to any one of 5 to 5.
  7. 腫瘍細胞に特異的に結合する抗体断片が、重鎖可変領域のアミノ酸配列として配列番号３に記載のアミノ酸配列を有し、軽鎖可変領域のアミノ酸配列として配列番号４に記載のアミノ酸配列を有する、Ｒｏｂｏ１に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）である、請求項１から６の何れかに記載の腫瘍の診断剤又は治療剤キット。 The antibody fragment that specifically binds to the tumor cell has the amino acid sequence shown in SEQ ID NO: 3 as the amino acid sequence of the heavy chain variable region, and the amino acid sequence shown in SEQ ID NO: 4 as the amino acid sequence of the light chain variable region The diagnostic or therapeutic agent kit for tumor according to any one of claims 1 to 6, which is a single-chain antibody fragment (scFv) that specifically binds to Robo1.
  8. 前記患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間が、前記第一の標的化分子の投与後６時間から１４４時間である、請求項１から７の何れかに記載の腫瘍の診断剤又は治療剤キット。 Sufficient time is allowed for the first targeting molecule adsorbed nonspecifically at the non-tumor site of the patient to be substantially excreted from the patient's blood after administration of the first targeting molecule. The diagnostic or therapeutic agent kit for a tumor according to any one of claims 1 to 7, which is 6 to 144 hours.
  9. 前記放射性同位元素が、64Ｃｕ、124Ｉ、76Ｂｒ、68Ｇａ、111Ｉｎ、99mＴｃ、67Ｇａ、123Ｉ、131Ｉ、または90Ｙである、請求項１から８の何れかに記載の腫瘍の診断剤又は治療剤キット。 9. The radioisotope according to claim 1, wherein the radioisotope is 64 Cu, 124 I, 76 Br, 68 Ga, 111 In, 99m Tc, 67 Ga, 123 I, 131 I, or 90 Y. Tumor diagnostic or therapeutic agent kit.
  10. 重鎖可変領域のアミノ酸配列として配列番号３に記載のアミノ酸配列を有し、軽鎖可変領域のアミノ酸配列として配列番号４に記載のアミノ酸配列を有する、Ｒｏｂｏ１に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）。 A single-chain antibody that specifically binds to Robo1, having the amino acid sequence set forth in SEQ ID NO: 3 as the amino acid sequence of the heavy chain variable region and the amino acid sequence set forth in SEQ ID NO: 4 as the amino acid sequence of the light chain variable region Fragment (scFv).
  11. 一対の親和性物質のうちの一方が連結されている、請求項１０に記載の一本鎖抗体フラグメント（ｓｃＦｖ）。 The single chain antibody fragment (scFv) according to claim 10, wherein one of the pair of affinity substances is linked.
  12. 請求項１０又は１１に記載の一本鎖抗体フラグメント（ｓｃＦｖ）をコードする核酸。 A nucleic acid encoding the single chain antibody fragment (scFv) according to claim 10 or 11.
  13. 腫瘍の診断又は治療を必要とする患者に、腫瘍細胞に特異的に結合する抗体断片と、一対の親和性物質のうちの一方とを連結した第一の標的化分子を投与し、前記第一の標的化分子が腫瘍部位に特異的に結合し、かつ当該腫瘍部位に安定して存在する間であって、前記患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間が経過した後に、放射性同位元素と、前記一対の親和性物質のうちの他方とを連結した第二の標的化分子を前記患者に投与し、前記放射性同位元素を前記腫瘍部位に局在化させることを含む、腫瘍の診断又は治療のための方法。 A patient in need of tumor diagnosis or treatment is administered with a first targeting molecule in which an antibody fragment that specifically binds to a tumor cell and one of a pair of affinity substances are linked, The first targeting molecule is non-specifically adsorbed to the non-tumor site of the patient while the targeting molecule specifically binds to the tumor site and is stably present at the tumor site. After a sufficient amount of time has passed for the patient to be substantially excreted from the blood of the patient, a second targeting molecule linking the radioisotope and the other of the pair of affinity substances is added to the patient. A method for diagnosing or treating a tumor, comprising administering to the tumor site and localizing the radioisotope to the tumor site.
  14. 一対の親和性物質のうちの一方が、ビオチン結合性タンパク質であり、一対の親和性物質のうちの他方がビオチン又はその誘導体である、請求項１３に記載の方法。 The method according to claim 13, wherein one of the pair of affinity substances is a biotin-binding protein and the other of the pair of affinity substances is biotin or a derivative thereof.
  15. ビオチン結合性タンパク質が、アビジン、ストレプトアビジン、タマビジン、又はそれらの誘導体である、請求項１４に記載の方法。 The method according to claim 14, wherein the biotin-binding protein is avidin, streptavidin, tamavidin, or a derivative thereof.
  16. 第二の標的化分子において、放射性同位元素と結合しうるキレート剤を用いて、放射性同位元素と前記一対の親和性物質のうちの他方とが連結している、請求項１３から１５の何れかに記載の方法。 The radioisotope and the other of the pair of affinity substances are linked to each other using a chelating agent capable of binding to the radioisotope in the second targeting molecule. The method described in 1.
  17. 腫瘍細胞に特異的に結合する抗体断片が、抗Ｒｏｂｏ１抗体である、請求項１３から１６の何れかに記載の方法。 The method according to any one of claims 13 to 16, wherein the antibody fragment that specifically binds to a tumor cell is an anti-Robo1 antibody.
  18. 第一の標的化分子が、腫瘍細胞に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）と、ストレプトアビジとを連結してなる４量体又は２量体の分子である、請求項１３から１７の何れかに記載の方法。 The first targeting molecule is a tetramer or dimer molecule formed by linking a single-chain antibody fragment (scFv) that specifically binds to a tumor cell and streptavidin. To 17. The method according to any one of 17 to 17.
  19. 腫瘍細胞に特異的に結合する抗体断片が、重鎖可変領域のアミノ酸配列として配列番号３に記載のアミノ酸配列を有し、軽鎖可変領域のアミノ酸配列として配列番号４に記載のアミノ酸配列を有する、Ｒｏｂｏ１に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）である、請求項１３から１８の何れかに記載の方法。 The antibody fragment that specifically binds to the tumor cell has the amino acid sequence shown in SEQ ID NO: 3 as the amino acid sequence of the heavy chain variable region, and the amino acid sequence shown in SEQ ID NO: 4 as the amino acid sequence of the light chain variable region The method according to any one of claims 13 to 18, which is a single-chain antibody fragment (scFv) that specifically binds to Robo1.
  20. 前記患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間が、前記第一の標的化分子の投与後６時間から１４４時間である、請求項１３から１９の何れかに記載の方法。 Sufficient time is allowed for the first targeting molecule adsorbed nonspecifically at the non-tumor site of the patient to be substantially excreted from the patient's blood after administration of the first targeting molecule. 20. A method according to any of claims 13 to 19, wherein the method is from 6 hours to 144 hours.
  21. 前記放射性同位元素が、64Ｃｕ、124Ｉ、76Ｂｒ、68Ｇａ、111Ｉｎ、99mＴｃ、67Ｇａ、123Ｉ、131Ｉ、または90Ｙである、請求項１３から２０の何れかに記載の方法。 The radioisotope, 64 Cu, which is 124 I, 76 Br, 68 Ga , 111 In, 99m Tc, 67 Ga, 123 I, 131 I or 90 Y,, according to any of claims 13 20 Method.
  22. 腫瘍の診断剤又は治療剤キットの製造のための、（ａ）腫瘍細胞に特異的に結合する抗体断片と、一対の親和性物質のうちの一方とを連結した第一の標的化分子、及び（ｂ）放射性同位元素と、前記一対の親和性物質のうちの他方とを連結した第二の標的化分子の使用であって、腫瘍の診断又は治療を必要とする患者に、前記第一の標的化分子を投与し、前記第一の標的化分子が腫瘍部位に特異的に結合し、かつ当該腫瘍部位に安定して存在する間であって、前記患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間が経過した後に、前記第二の標的化分子を前記患者に投与し、前記放射性同位元素を前記腫瘍部位に局在化させることを特徴とする、上記の使用。 (A) a first targeting molecule in which an antibody fragment that specifically binds to a tumor cell and one of a pair of affinity substances are linked for the production of a tumor diagnostic or therapeutic agent kit; and (B) use of a second targeting molecule in which a radioisotope is linked to the other of the pair of affinity substances, wherein the first A targeting molecule is administered, while the first targeting molecule specifically binds to a tumor site and is stably present at the tumor site, non-specifically at a non-tumor site of the patient After a sufficient time has elapsed for the adsorbed first targeting molecule to be substantially excreted from the patient's blood, the second targeting molecule is administered to the patient and the radioisotope Use as described above, characterized in that is localized at the tumor site.
  23. 一対の親和性物質のうちの一方が、ビオチン結合性タンパク質であり、一対の親和性物質のうちの他方がビオチン又はその誘導体である、請求項２２に記載の使用。 The use according to claim 22, wherein one of the pair of affinity substances is a biotin-binding protein and the other of the pair of affinity substances is biotin or a derivative thereof.
  24. ビオチン結合性タンパク質が、アビジン、ストレプトアビジン、タマビジン、又はそれらの誘導体である、請求項２３に記載の使用。 The use according to claim 23, wherein the biotin-binding protein is avidin, streptavidin, tamavidin, or a derivative thereof.
  25. 第二の標的化分子において、放射性同位元素と結合しうるキレート剤を用いて、放射性同位元素と前記一対の親和性物質のうちの他方とが連結している、請求項２２から２４の何れかに記載の使用。 25. The method according to any one of claims 22 to 24, wherein in the second targeting molecule, the radioisotope is linked to the other of the pair of affinity substances using a chelating agent capable of binding to the radioisotope. Use as described in.
  26. 腫瘍細胞に特異的に結合する抗体断片が、抗Ｒｏｂｏ１抗体である、請求項２２から２５の何れかに記載の使用。 26. The use according to any one of claims 22 to 25, wherein the antibody fragment that specifically binds to a tumor cell is an anti-Robo1 antibody.
  27. 第一の標的化分子が、腫瘍細胞に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）と、ストレプトアビジとを連結してなる４量体又は２量体の分子である、請求項２２から２６の何れかに記載の使用。 The first targeting molecule is a tetramer or dimer molecule obtained by linking a single-chain antibody fragment (scFv) that specifically binds to a tumor cell and streptavidin. 27. Use according to any one of.
  28. 腫瘍細胞に特異的に結合する抗体断片が、重鎖可変領域のアミノ酸配列として配列番号３に記載のアミノ酸配列を有し、軽鎖可変領域のアミノ酸配列として配列番号４に記載のアミノ酸配列を有する、Ｒｏｂｏ１に特異的に結合する一本鎖抗体フラグメント（ｓｃＦｖ）である、請求項２２から２７の何れかに記載の使用。 The antibody fragment that specifically binds to the tumor cell has the amino acid sequence shown in SEQ ID NO: 3 as the amino acid sequence of the heavy chain variable region, and the amino acid sequence shown in SEQ ID NO: 4 as the amino acid sequence of the light chain variable region 28. Use according to any of claims 22 to 27, which is a single chain antibody fragment (scFv) that specifically binds to Robo1.
  29. 前記患者の非腫瘍部位に非特異的に吸着された前記第一の標的化分子が患者の血中から実質的に排出されるのに十分な時間が、前記第一の標的化分子の投与後６時間から１４４時間である、請求項２２から２８の何れかに記載の使用。 Sufficient time is allowed for the first targeting molecule adsorbed non-specifically to the non-tumor site of the patient to be substantially excreted from the patient's blood after administration of the first targeting molecule. 29. Use according to any of claims 22 to 28, which is 6 hours to 144 hours.
  30. 前記放射性同位元素が、64Ｃｕ、124Ｉ、76Ｂｒ、68Ｇａ、111Ｉｎ、99mＴｃ、67Ｇａ、123Ｉ、131Ｉ、または90Ｙである、請求項２２から２９の何れかに記載の使用。 The radioisotope, 64 Cu, which is 124 I, 76 Br, 68 Ga , 111 In, 99m Tc, 67 Ga, 123 I, 131 I or 90 Y,, according to any of claims 22 29 use.
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