Drug – bio-affecting and body treating compositions – Radionuclide or intended radionuclide containing; adjuvant... – In an organic compound
Patent
1994-06-16
1998-06-09
Kight, John
Drug, bio-affecting and body treating compositions
Radionuclide or intended radionuclide containing; adjuvant...
In an organic compound
424 177, 424 111, 424 169, 530300, A61K 5100, A61M 3614
Patent
active
057629064
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
This invention relates to improvements in the labelling of monoclonal antibodies and other proteins with .sup.32 P. The term "protein" as used herein includes polypeptides.
Radiation therapy, particularly using .sup.32 P, is of interest as a possible method of treatment of certain cancer conditions and it is therefore of interest to be able to label antibodies or other targeting molecules with .sup.32 P. However, the labelling of the antibody or similar targeting molecule must be done in such a way that the specificity of the antibody or similar targeting molecule is retained in a labelled molecule that has appropriate in vivo stability.
Our earlier Patent, GB-B-2,186,579, describes a system for modifying a protein that will bind with a tumour-associated structure comprising the introduction into the binding protein of a peptide region which is capable of acting as a substrate for a phosphokinase. The resulting modified binding protein can then be .sup.32 P labelled by reacting it with a .sup.32 P labelled gamma nucleotide triphosphate in the presence of a phosphokinase.
In accordance with the procedures described in our earlier Patent GB-B-2,186,579, the peptide region capable of acting as a substrate for a phosphokinase is introduced into the binding protein by using a hetero-bifunctional protein crosslinking agent. For example, the targeting molecule may be reacted with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) followed by reduction with dithiothreitol. The reaction with SPDP introduces the dithiopropionyl group onto a side-chain amino group of a lysine residue in the targeting molecule while the subsequent reduction step converts the dithio grouping into a terminal thiol group. This terminal thiol group provides the reactive site for introduction of the substrate molecule.
In accordance with the procedure described in our earlier UK Patent, it is then necessary to activate the substrate molecule by reacting the N-terminus of the substrate molecule with a bridging molecule which in turn can be reacted with the terminal thiol group on the targeting molecule. For example, if the substrate molecule has an N-terminal leucine residue, its amino group can be reacted with an N-hydroxy-succinimidyl ester to give for example an iodoacetamide or a phenyl maleimide which can then react with the thiol group of the thiopropionamido residue introduced on the targeting molecule so that the substrate molecule becomes attached to the targeting molecule through a short bridging group including a thio link. If the substrate molecule has an N-terminal cysteine residue, then its thiol group can be reacted with succinimidyl-4-(p-maleimidophenyl)butyrate and the reaction product then reacted with the targeting molecule.
In the targeting molecule/substrate conjugates described in our earlier Patent, the substrate molecule is linked through its N-terminus by a short sulphur-containing bridge to the terminal amino group of the lysine residues of the targeting molecule. However, the procedure to bring about this conjugation is chemically complex involving the use of the hetero-bifunctional bridging molecules.
DESCRIPTION OF THE INVENTION
We have now found that it is possible to introduce a targeting molecule/substrate conjugate by much simpler synthetic methods involving conventional peptide chemistry to give a phosphorylatable conjugate in which the substrate molecule is directly bonded through its carboxy terminus through a peptide link to the targeting molecule and that the resulting targeting molecule/substrate conjugates hereinafter referred to as structurally-modified targeting proteins, structurally-modified proteins or targeting protein/substrate peptide conjugates, when labelled with .sup.32 P, give conjugates that are more effective in vivo as therapeutic reagents.
Accordingly, the present invention provides a structurally-modified protein that will bind to a tumour-associated structure wherein the amino group in at least one basic amino acid in the binding protein is structural
REFERENCES:
patent: 5459240 (1995-10-01), Foxwell et al.
Foxwell et al (1988), British Journal of Cancer, vol. 57, pp. 489-493 Conjugation of Monoclonal Antibodies to a Synthetic Peptide Substrate for Protein Kinase: A Method for Labeling Antibodies with 32p.
Sullivan and Wong (1991), Analytical Biochemistry, vol. 197, pp. 65-68. A Manual Sequencing Method for Identification of Phosphorylated Amino Acids in Phospho Peptides.
Carlsson et al (1978), Biochem. Journal, vol. 173, pp. 723-737. Protein Thiolation and Reversible Protein--Protein Conjugation.
Zuhay (1983), Biochemistry (Protein Structure and Function), pp. 3-7.
Bramson et al (1985), J. Biological Chemistry, vol. 260, No. 29, pp. 15452-15457, "The Use of N-methylated Peptides and Depsipeptides to Probe the Binding of Heptapeptide Substrates to Camp-Dependent Protein Kinase".
Thomas et al (1987), Biochemistry, vol. 26, pp. 4461-4466, "Role of Enzyme-Peptide Substrate Backbone Hydrogen Bonding in Determining Protein Kinase Substrate Specificties".
Chemical Abstracts vol. 113, No. 3, 16 Jul. 1990, Columbus, Ohio USA, #20373s; Jenkins et al "Meaurement of protein . . . ", p. 315.
Biological Abstracts, vol. 86, No. 7, 1988, Philadelphia, PA, USA, #71619. Foxwell et al, "Conjugation of monoclonal antibodies . . . ".
British Technology Group Ltd.
Jones Dameron
Kight John
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