Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• radioimmunoconjugate may be used as a chemotherapy drug to kill cancerous tissues. There is a need for improved methods for the production of radioimmunoconjugates. This invention addresses this need.
• the conjugation reaction mixture comprises a bicarbonate buffer. In another embodiment, the conjugation reaction mixture comprises a phosphate buffer. In still a further embodiment, the conjugation reaction mixture has a pH of about 8.0 to about 9.2.
• the filtering is performed in a HEPES buffer. In another embodiment, the filtering is performed in a NaAc buffer. In still a further embodiment, the filtering comprises a molecular weight cut off at least about 10,000 Da, at least about 20,000 Da, or at least about 40,000 Da.
• the chelation reaction mixture comprises gentisic acid or ascorbic acid.
• the biological molecule is an antibody, an antigen- binding fragment thereof, a single-chain protein comprising the antigen-binding polypeptide sequences of an antibody, a single-domain antibody, an analog of any of the foregoing, or a derivative of any of the foregoing.
• the antigen binding fragment is a monoclonal antibody variable region.
• the biological molecule is a protein comprising an antigen binding sequence of an antibody.
• the biological molecule is a naturally, synthetically, or recombinantly produced protein comprising an antigen binding sequence of an antibody that binds an antigen on the surface of a cell.
• the antigen on the surface of the target cell is CD-33.
• the biological molecule is HuM 195.
• the methods described herein relate to a method for producing an [Ac-225]-p-SCN-Bn-DOTA/HuM195 radioimmunoconjugate, the method comprising the steps of: (a) conjugating p-SCN-Bn-DOTA to a HuM 195 antibody in a conjugation reaction mixture comprising a bicarbonate buffer and having a pH of about 8.0 to about 9.2, for about 1.5 hours at about 37oC to generate a p-SCN-Bn-DOTA/HuM195 immunoconjugate, (b) filtering the conjugation reaction mixture through a filter having a molecular weight cut off at least about 10,000 Da, at least about 20,000 Da, or at least about 40,000 Da so as to purify the p p-SCN-Bn-DOTA/HuM195 immunoconjugate, wherein the filtering is performed with a HEPES buffer or NaAc buffer, (c) chelating one or more actinium-225 radionuclides with
• the methods described herein further comprise purifying radionconjugate or the [Ac-225]-p-SCN-Bn-DOTA/HuM195
• Figure 6 SE - HPLC/rad. chromatogram of the conjugate (main protein fraction after purification) labelled with Ac-225.
• the methods described herein relate to a one step chelation post conjugation process for the manufacture of a radioimmunoconjugate.
• the radioimmunoconjugates described herein can be prepared by first forming a conjugated targeting moiety and then chelating a radionuclide with the conjugated targeting moiety to form a radioimmunoconjugate.
• the conjugated targeting moiety may be radiolabeled at any time following conjugation to the targeting moiety.
• a mixture of HuM- 195 and 225 Ac solution in acetate buffer having pH of about 5.5 to about 7.0 is incubated for about 80 to about 90 minutes in the presence of small quantities of a free radical scavenger.
• the labelling yield can be determined by instant thin layer chromatography (ITLC).
• the sample mixture can then purified by gravity size exclusion chromatography (SEC).
• SEC gravity size exclusion chromatography
• the amount Of 225 Ac and protein can be measured by non-destructive high resolution gamma spectrometry and UV spectrophotometry, respectively.
• radiopharmaceutical may be used as an imaging contrast agent to produce PET or other radiographic images.
• the radiopharmaceutical may be used as a therapeutic agent that delivers doses of radiation to specific structures or sites of physiological activity in the body.
• One skilled in the art will appreciate other pharmacological uses of the
• targeting moiety refers to any protein, antibody, antibody fragment, hormone, peptide, growth factor, antigen, hapten or any other carrier which functions to recognize a specific biological target site.
• Antibody and antibody fragment refers to any polyclonal, monoclonal, chimeric, human, mammalian, single chains, dimeric and tetrameric antibody or antibody fragment. Such biological carrier, when attached to a functionalized complex, serves to carry the attached radionuclide to specific targeted tissues.
• antibody refers to any polyclonal, monoclonal, chimeric antibody or heteroantibody.
• the yield of the conjugated targeting moiety or the radioimmunoconjugate is at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the final product.
• the Fab' regions can be derived from antibodies of animal (especially mouse or rat) or human origin or may be chimeric (Morrison et al., Proc Natl. Acad. Sci. USA 81, 6851-6855 (1984)) or humanized (Jones et al., Nature 321, 522-525 (1986), and published UK patent application No. 8707252).
• phage and other antibody display methods afford the opportunity to manipulate selection against the antigen target in vitro and without the limitation of the possibility of host effects on the antigen or vice versa.
• variable region of a light chain VL and the variable region of a heavy chain optionally linked by a flexible linker, such as a polypeptide sequence, in either VL-linker-VH orientation or in VH-linker-VL orientation.
• a single chain Fv fragment can be produced by linking two variable domains via a disulfide linkage between two cysteine residues.
• Antibodies may be directly linked to each other via a chemical cross linking agent or can be connected via a linker sequence (e.g., a peptide sequence) to form multimers.
• a linker sequence e.g., a peptide sequence
• salt means any salt of a compound of formula I which is sufficiently non-toxic to be useful in therapy of mammals.
• Representative of those salts, which are formed by standard reactions, from both organic and inorganic sources include, for example, sulfuric, hydrochloric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, palmoic, mucic, glutamic, d-camphoric, glutaric, glycolic, phthalic, tartaric, formic, lauric, steric, salicylic, methanesulfonic, bensenesulfonic, sorbic, picric, benzoic, cinnamic and other suitable acids.
• the term "therapeutically effective amount” means an amount of a radioimmunoconjugate that produces a therapeutic effect on the disease treated.
• the therapeutically effective amount will vary depending on the mammal, the
• the radioimmunoconjugate pharmaceutical compositions can be administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally,
• Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
• a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
• antibacterial agents such as benzyl alcohol or methyl parabens
• antioxidants such as as
• the clearing agent binds to the radioimmunoconjugate in a monovalent manner, while its appended glycosyl residues direct the entire complex to the liver, where rapid metabolism takes place. Clearing agents are discussed in greater detail in U.S. Ser. Nos. 09/314,135 and 09/337,756.
• BFC Bifunctional chelating agents
• HuM-195 Anti-CD33 antibody construct for therapy of myeloid leukemia.
• HuM- 195 is a recombinant IgGl mAb combining CDR regions of murine M- 195 with human framework/constant regions.
• ITLC Instant thin layer chromatography
• Reaction vial and heating block [00228] 3.3.2.1 Take a new reaction tube (e.g. 2 rnL capacity Eppendorf tube) and wash it with a few mLs of 0.1 M HCl, water for injection H2O and finally with the 0.5 M NaAc (0.5 M NH4Ac) buffer.
• a new reaction tube e.g. 2 rnL capacity Eppendorf tube
• wash it with a few mLs of 0.1 M HCl, water for injection H2O and finally with the 0.5 M NaAc (0.5 M NH4Ac) buffer.
• Solution of gentisic acid in 3 M NaAc Weigh 0.033 g of gentisic acid in dissolve in 1 mL 3 M NaAc. Before use, filter the solution through a 0.45 ⁇ m cellulose acetate membrane filter. Keep the solution in a 2 mL - capacity Eppendorf vial in cool and darkness.
• 150 g/L 1-AA solution Weigh 1.5 g of AA and dissolve in 10 mL free metal water. Filter the solution through a 0.45 ⁇ m cellulose acetate membrane filter. Keep the solution in a 25 mL - HDPE container in cool and darkness.
• 50 niM DTPA solution Weigh 0.49 g of DTPA and dissolve in 25 mL free metal water. Filter the solution through a 0.45 ⁇ m cellulose acetate membrane filter. Keep the solution in a 25 mL - HDPE container
• pH papers thin strips of pH paper in the range 4 - 10
• R c radiochemical purity of the labelled conjugate in %
• I c area measured under the conjugate peak on the
• I t total area measured on the SE - HPLC/rad.
• a H percentage of Ac - 225 associated with high MW aggregates
• % I h area measured on the left side of the conjugate peak
• a L percentage of Ac - 225 associated with low MW aggregates
• % I t area measured on the right side of the conjugate peak until the

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• 2010
  • 2010-07-22 CA CA2768658A patent/CA2768658C/en active Active
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