WO1991002547A1 - Anticorps radiomarques d'imagerie - Google Patents

Anticorps radiomarques d'imagerie Download PDF

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Publication number
WO1991002547A1
WO1991002547A1 PCT/AU1990/000372 AU9000372W WO9102547A1 WO 1991002547 A1 WO1991002547 A1 WO 1991002547A1 AU 9000372 W AU9000372 W AU 9000372W WO 9102547 A1 WO9102547 A1 WO 9102547A1
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WO
WIPO (PCT)
Prior art keywords
fragment
antibody
thiolated
fab
labelled
Prior art date
Application number
PCT/AU1990/000372
Other languages
English (en)
Inventor
Fook-Thean Lee
Graeme Boniface
Original Assignee
Australian Nuclear Science & Technology Organisation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Australian Nuclear Science & Technology Organisation filed Critical Australian Nuclear Science & Technology Organisation
Publication of WO1991002547A1 publication Critical patent/WO1991002547A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1282—Devices used in vivo and carrying the radioactive therapeutic or diagnostic agent, therapeutic or in vivo diagnostic kits, stents
    • 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
    • A61K51/1018—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 material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo

Definitions

  • the present invention relates to radio-labelled antibodies or other proteinaceous materials for imaging.
  • the present invention is concerned with scintigraphic detection of thrombi in mammals including humans.
  • substances and processes developed to facilitate detection of thrombi may also have usefulness for other imaging such as of tumours.
  • a method of producing a kit for scintigraphic detection of thrombi in mammals including humans comprising taking a material which is directed specifically against blood clots, the material being from the group consisting of a proteinaceous material, a monoclonal antibody, a single domain antibody, or an epitope binding fragment of a monoclonal antibody or a single domain antibody, and conjugating the material with a thiolating agent, whereby there is provided a conjugated material adapted to be labelled with an acceptable radionuclide.
  • the monoclonal antibody is Mab 3 B6/22 (3B6) .
  • Monoclonal antibody 3B6 recognises the D-dimer (DD) epitope of human cross-linked fibrin.
  • Monoclonal antibody 3B6 is available from AGEN Biomedical Limited, of Brisbane, Queensland, Australia amd is described in Australian patent no. 572,125.
  • a preferred radionuclide is "' ⁇ c (technetium-99m) because of various advantageous physical properties such as being a pure gamma emitter of 140 keV, short half life, and being readily available.
  • other radionuclides might be useable such as rhenium.
  • Rhenium has several isotopic forms namely 186, 188, 189 and 191 and is a beta and gamma emitter and similarity of chemical characteristics with technetium-99m makes it a candidate for use in conjugating to protein carriers. Rhenium-protein conjugates may be useful for therapeutic applications.
  • the present invention can be advantageously implemented where use is made of the Fab' fragment of a suitable monoclonal antibody.
  • the Fab' fragment can be produced by (i) pepsin digestion of the antibody to produce the F(ab')_ fragment and then (ii) this fragment is reduced by a suitable reducing agent such as dithiothreitol (DTT) to produce the Fab' fragment.
  • DTT dithiothreitol
  • the smaller fragment Fab' can be obtained but it is now pointed out that this is a reversible process.
  • the present invention makes use of a thiolating agent (with or without the preliminary step of reduction with an agent such as DTT) and most significantly it has been found that the use of the thiolating agent is especially beneficial in suppressing or preventing Fab' fragments recombining to form F(ab')_. Furthermore enhanced labelling efficiency was found to occur by using the thiolated Fab' fragments of the antibody. Furthermore it has been found that an initial reduction step with an agent such as DTT is unnecessary.
  • a preferred embodiment of the invention consists in using the Fab' fragment of the antibody due to enhanced labelling
  • the invention also extends to labelling the antibody and other fragments thereof including F(ab')_.
  • F(ab')_ When dealing with problems of the vascular system such as thrombolic disorders, it is believed the Fab' fragment will give the best results for scintigraphy.
  • the Fab* fragment has a relatively small size which allows clot penetration together with rapid blood clearance and this will provide an excellent target-to-blood ratio well suited for scintigraphic detection.
  • the thiolating agent DL N-Acetylhomocystein-thiolactone is used.
  • Preferred embodiments of the invention may include exchange labelling of the thiolated antibody or reduced fragment, for example by using radionuclide labelled gluconate and preferably a purification step follows, for example by the use of gel column chromatography or high pressure liquid chromatography (HPLC) . The resultant product is then ready for injection.
  • An advantageous embodiment of the invention produces a three vial kit ready for use with radionuclide labelling which takes place just before use.
  • the vials are produced as follows:
  • a F(ab*) 2 fragment of a monoclonal antibody specific to thrombi is prepared by a known method.
  • the fragment is thiolated to produced the Fab' fragment and purification takes place to provide a source of thiolated fragment which can then be freezed dried and stablised into the vial.
  • Thiolation is best carried out with the use of a suitable catalyst.
  • Thiolation of proteins and specifically antibodies is known, as is the use of catalyst (see for example Warzynski et al, J. Immunol Methods 35, 157-168, 1980) . Routine experimentation is used to determine the precise process conditions to suit the particular antibodies.
  • a vial of a suitable buffer is provided for adding to the freeze dried thiolated fragment prior to labelling.
  • a suitable renal or hepatic imaging agent such as Sn-gluconate to provide a liga ⁇ d which will take up technetium-99m usually supplied in pertechnetate form.
  • Suitable agents include glucohephonate, MDP, pyrophosphate and HIDA derivatives.
  • the kit is used by adding the sterile buffer from the second vial to the antibody vial.
  • Pertechnetate is added to the imaging agent vial and a suitable volume of this material is then added to the antibody vial.
  • the mixture is typically incubated for five to ten minutes allowing for quatitative transfer of technetium-99m from the imaging kit to the antibody.
  • the resultant technetium-99m labelled antibodies are ready for injection into patients without further purification.
  • Use of the present invention permits a simple, readily controlled chemical reaction to be used for producing the kit and the kit is relatively simple to use in practice.
  • a high specific radioactivity technetium-labelled Fab' on a weight-for-weight basis is obtainable.
  • the invention extends to a kit for use in scintigraphic imaging of thrombi in mammals comprising the thiolated material produced in the method described in any one of the forms above and a supply of exchange complex in a form suitable for labelling with a radionuclide, the kit being in a form such that reaction of the thiolated material with the exchange complex (when labelled) produces a solution for injection into a mammal with or without further purification.
  • the invention consists in a method of scintigraphic imaging comprising using a kit as described above.
  • a kit as described above.
  • the antifibrin monoclonal antibody DD-3B6/22 and its F(ab') 2 fragment and fibrin D dimer were supplied by Agen Biomedical Pty. Ltd. (Brisbane, Australia) .
  • Dithiothreitol and immnoglobulins free bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St Louis).
  • RM 6 a renal imaging kit consisting of calcium gluconate, stannous chloride and Tc99m pertechnetate were obtained from Australian Radioisotopes (Sydney, Australia) .
  • Biogel P-6DG was from Biorad.
  • Sepharose 6 MB was purchased from Pharmacia (Uppsala, Sweden) .
  • Renal imaging agent RM6 was constituted with 1.0ml of pertechnetate eluted from a technetium generator having radioactivity in the range 30 to 300 mCi/ml. A 0.1ml aliquot of this technetium-99m mixture was added to the thiolated antibody.
  • the mixture having a final protein concentration of 0.95 ⁇ g/ml was incubated at about 37°C. By known monitoring techniques, it was found that quantitative labelling (> 99%) of antibody can be achieved in under 15 minutes.
  • Antifibrin monoclonal antibody DD-3B6/22 was subjected to pepsin digestion to produce the F(ab')_ fragment.
  • Dithiothreitol reduction was effected to produce the Fab' fragment, a reducing agent: antibody mole ratio of 12:1 being used to permit high radionuclide incorporation in the subsequent step.
  • 1.0 mg of the fragment F(ab') 2 in phosphate buffer saline was incubated with 12.0 ⁇ l of a 10 mM solution of dithiothreitol in a final volume of 300 ⁇ l at 37 deg C for 30 mins.
  • Excess reducing agent was removed by centrifugal desalting using Biogel P-6DG equilibrated with PBS.
  • the reduced antibody was obtained in an undiluted form and used immediately. Without being bound to any particular theory, the inventors suggest that this may be due to reduction of disulphide bridges around the antibody hinge region.
  • Technetium-99m ligand complex was prepared by adding 2.5 ⁇ l of a mixture containing 20 ⁇ g of calcium gluconate and 0.5 ⁇ g of stannous chloride to 250 ⁇ l of pertechnetate. 5. The reduced and thiolated antibody fragment was incubated with 120 ⁇ l of this Tc99m/gluconate mixture for 10 mins at room temperature. Excess technetium-99m was removed by centrifugal desalting.
  • the mixture was thiolated by the addition of 0.4ml 2-pyridinealdoxine methiodide and 0.4ml N-acetylhomocysteine thiolactone and the ph adjusted to 9.0. 3. The mixture was incubated on ice for two hours while maintaining the ph at 9.0.
  • the antibody mixture was purified by centrifugal desalting on Biogel P-6DG equilibrated in deaerated.water to produce purified Fab' fragment. 5. The fragment was divided in 0.7mg lots and lyophilised and upon completion of freezed drying in vials, the vials were sealed in vacuum and stored at -20°C.
  • the freezed dried thiolated Fab' fragment was labelled with the first step comprising adding 0.3ml of a 0.1M solution of sodium acetate buffer at ph 5.6 to the fragment. 7. 0.3ml of technetium-99m gluconate was added to the fragment and the result mixture incubated at up to 37°C.
  • SAP serum amyloid proteins
  • N-acetylhornocysteine thiolactone (0.25M). The mixture was kept at 0- °C overnight.
  • the antibody can be desalted in a column equilibrated with deoxygenated water instead of PBS.
  • Another alternative is to use gel column chromatography for purification of the antibody instead of HPLC.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Dispersion Chemistry (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Immunology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

On peut procéder à la détection scintigraphique de caillots sanguins par injection d'une solution d'un agent radiomarqué pouvant être une matière provenant d'un kit lyophilisé reconstitué et pouvant être ensuite marqué par exemple à l'aide de technétium-99m. L'agent est produit à partir d'une matière de départ provenant du groupe composé de matières protéiques, d'anticorps monoclonaux, d'anticorps à un seul domaine ou d'un fragment de liaison d'épitopes d'anticorps monoclonaux ou d'anticorps à un seul domaine. On a choisi la matière de départ pour qu'elle soit spécifiquement dirigée contre les caillots sanguins, le passage au thiol étant effectué, par exemple, à l'aide de DL N-acéthylhomocystéine-thiolactone. Selon un mode de réalisation important l'étape de passage au thiol produit le fragment Fab'.
PCT/AU1990/000372 1989-08-24 1990-08-24 Anticorps radiomarques d'imagerie WO1991002547A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPJ596089 1989-08-24
AUPJ5960 1989-08-24

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WO1991002547A1 true WO1991002547A1 (fr) 1991-03-07

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EP (1) EP0489061A4 (fr)
CA (1) CA2065362A1 (fr)
WO (1) WO1991002547A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5346687A (en) * 1989-08-09 1994-09-13 Rhomed Incorporated Direct radiolabeling of antibody against stage specific embryonic antigen for diagnostic imaging
US5460785A (en) * 1989-08-09 1995-10-24 Rhomed Incorporated Direct labeling of antibodies and other protein with metal ions
US5879657A (en) * 1993-03-30 1999-03-09 The Dupont Merck Pharmaceutical Company Radiolabeled platelet GPIIb/IIIa receptor antagonists as imaging agents for the diagnosis of thromboembolic disorders
WO1999012579A1 (fr) * 1997-09-08 1999-03-18 The General Hospital Corporation Agents d'imagerie destines a la detection et au controle d'une plaque cardiovasculaire
WO2003000736A1 (fr) * 2001-06-26 2003-01-03 Agen Biomedical Limited Anticorps humanises derives de dd-3b6/22, specifiques du fragment d-dimere de la fibrine
EP1442302A2 (fr) * 2001-10-12 2004-08-04 Molecular Probes, Inc. Complexes d'anticorps et procedes d'immunomarquage
US7060251B1 (en) 1997-09-08 2006-06-13 The General Hospital Corporation Imaging agents for early detection and monitoring of cardiovascular plaque
CN106324251A (zh) * 2016-08-08 2017-01-11 上海睿康生物科技有限公司 小片段BMG抗体的制备方法及β2‑微球蛋白检测试剂盒

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4434151A (en) * 1982-11-08 1984-02-28 Medi-Physics, Inc. Bifunctional chelating agents
WO1988007382A2 (fr) * 1987-04-02 1988-10-06 Centocor Cardiovascular Imaging Partners, L.P. Procede de marquage d'anticorps a l'aide d'un ion metallique
AU2068588A (en) * 1987-08-12 1989-02-16 Immunomedics Inc. Preparation of radiolabeled conjugates
EP0318948A2 (fr) * 1987-12-02 1989-06-07 Neorx Corporation Immunoconjugués clivables pour la délivrance et libération d'agents sous forme naturelle
WO1989007456A1 (fr) * 1988-02-09 1989-08-24 Mallinckrodt, Inc. Procede de preparation d'une proteine marquee par un nucleide radio-metallique
WO1989009405A1 (fr) * 1988-04-01 1989-10-05 Immunomedics, Inc. Procede de radiomarquage des proteines
WO1990005544A1 (fr) * 1988-11-21 1990-05-31 Centocor, Inc. Procede d'imagerie de thrombi coronaires

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU572125B2 (en) * 1983-03-17 1988-05-05 Mabco Limited Monoclonal antibodies with specificity for crosslinked fibrin and their diagnotic uses

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4434151A (en) * 1982-11-08 1984-02-28 Medi-Physics, Inc. Bifunctional chelating agents
WO1988007382A2 (fr) * 1987-04-02 1988-10-06 Centocor Cardiovascular Imaging Partners, L.P. Procede de marquage d'anticorps a l'aide d'un ion metallique
AU2068588A (en) * 1987-08-12 1989-02-16 Immunomedics Inc. Preparation of radiolabeled conjugates
EP0318948A2 (fr) * 1987-12-02 1989-06-07 Neorx Corporation Immunoconjugués clivables pour la délivrance et libération d'agents sous forme naturelle
WO1989007456A1 (fr) * 1988-02-09 1989-08-24 Mallinckrodt, Inc. Procede de preparation d'une proteine marquee par un nucleide radio-metallique
WO1989009405A1 (fr) * 1988-04-01 1989-10-05 Immunomedics, Inc. Procede de radiomarquage des proteines
WO1990005544A1 (fr) * 1988-11-21 1990-05-31 Centocor, Inc. Procede d'imagerie de thrombi coronaires

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0489061A4 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5460785A (en) * 1989-08-09 1995-10-24 Rhomed Incorporated Direct labeling of antibodies and other protein with metal ions
US5861139A (en) * 1989-08-09 1999-01-19 Rhodes; Buck A. Direct labeling of peptides with metal ions
US5346687A (en) * 1989-08-09 1994-09-13 Rhomed Incorporated Direct radiolabeling of antibody against stage specific embryonic antigen for diagnostic imaging
US5879657A (en) * 1993-03-30 1999-03-09 The Dupont Merck Pharmaceutical Company Radiolabeled platelet GPIIb/IIIa receptor antagonists as imaging agents for the diagnosis of thromboembolic disorders
US6022523A (en) * 1993-03-30 2000-02-08 Dupont Pharmaceuticals Company Radiolabeled platelet GPIIb/IIIa receptor antagonists as imaging agents for the diagnosis of thromboembolic disorders
US7060251B1 (en) 1997-09-08 2006-06-13 The General Hospital Corporation Imaging agents for early detection and monitoring of cardiovascular plaque
WO1999012579A1 (fr) * 1997-09-08 1999-03-18 The General Hospital Corporation Agents d'imagerie destines a la detection et au controle d'une plaque cardiovasculaire
WO2003000736A1 (fr) * 2001-06-26 2003-01-03 Agen Biomedical Limited Anticorps humanises derives de dd-3b6/22, specifiques du fragment d-dimere de la fibrine
US7087724B2 (en) 2001-06-26 2006-08-08 Agen Biomedical Ltd Carrier molecules
US7459143B2 (en) 2001-06-26 2008-12-02 Agen Biomedical Ltd. Carrier molecules
EP1442302A2 (fr) * 2001-10-12 2004-08-04 Molecular Probes, Inc. Complexes d'anticorps et procedes d'immunomarquage
EP1442302A4 (fr) * 2001-10-12 2007-03-28 Molecular Probes Inc Complexes d'anticorps et procedes d'immunomarquage
CN106324251A (zh) * 2016-08-08 2017-01-11 上海睿康生物科技有限公司 小片段BMG抗体的制备方法及β2‑微球蛋白检测试剂盒
CN106324251B (zh) * 2016-08-08 2019-03-29 上海睿康生物科技有限公司 小片段BMG抗体的制备方法及β2-微球蛋白检测试剂盒

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Publication number Publication date
EP0489061A1 (fr) 1992-06-10
EP0489061A4 (en) 1992-10-14
CA2065362A1 (fr) 1991-02-25

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