Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 6 to 7 amino acid residues in defined sequence
Reexamination Certificate
1996-09-20
2001-09-18
Scheiner, Toni R. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
6 to 7 amino acid residues in defined sequence
C530S328000, C530S326000, C530S327000, C530S333000, C530S391700, C424S184100, C424S178100, C424S009100
Reexamination Certificate
active
06291639
ABSTRACT:
DESCRIPTION
The object of this invention are metal-complexing, cysteine-free peptides that may be coupled, either directly or through a linker, with an organ-specific probe, and thus accumulate specifically as a conjugate in tumours, organs, tissues, or focuses of inflammation, said organ-specific probes being, for example, antibodies or partial antibody sequences used against tumour-related antigens such as the carcinoembryonal antigen (CIA), which thus accumulate specifically in tumours.
This invention further relates to methods for producing the metal-complexing cysteine-free peptides as well as their conjugates. The present invention further relates to the use of said conjugates as ingredients of a kit for in-vivo diagnostics or in-vivo therapy, and radio-pharmaceuticals that contain said conjugates and radionuclides. The organ-specific conjugates are used for the visualization of tumours, organs, or focuses of inflammation.
Apart from cardiovascular diseases, malignant carcinomas are a frequent cause of death due to their uncontrolled growth; even if the primary tumour has been removed, metastases that may be very small cannot be localized and thus not be removed. Therapeutical checks after operations or chemotherapy include, on the one hand, imaging methods such as CT or MR, on the other, determination of the content of tumour-related antigens such as CEA in the patient's serum, especially with colorectal tumours.
It is a disadvantage of these imaging methods that they cannot distinguish between the cicatrix and local recurrence. Where the CEA content in the serum is determined, the disadvantage, besides lacking tumour localization, is low sensitivity. On the one hand, formation of metastases cannot be excluded even with CEA values in the normal range, on the other hand non-malignant diseases such as inflammatory processes may result in increased CEA values.
Therefore early localization of tumours, or a tumour-specific treatment, could be decisive for the success of a therapy, especially when the diagnosis is not safe. This specific approach is facilitated by immuno-scintigraphy, an imaging method applied in nuclear medicine that is characterized by using isotope-labelled antibodies or antibody structures.
The radionuclides that are most frequently used in nuclear-medical diagnostics today are I-131, I-123, In-111 and Tc-99m; they exist in covalent bonding (I-131, I-123) or as a complex compound (In-111, Tc-99m). Although the radionuclides listed here minimize exposure of the patient to radiation due to their low half-lives, I-131, I-123 and In-111 have several disadvantages. Thus I-131, having a &ggr;-energy of 364 keV and a half-life of 8 d, emanates additional &bgr;-radiation which is highly tissue-damaging. Studies by various research teams on In-111 having &ggr;-energies of 172 keV and 245 keV and a half-life of 2.8 d, proved that organ-specific substances labelled with In-111 result in high concentrations in the reticuloendothelial system, especially in the liver, which makes hepatic diagnostics difficult if not impossible (Fairweather et al., Br. Med. J., 287, 167-170, 1983; Hnatowich et al., J. Nucl. Med., 26, 849-858, 1985). With its half-life of 13.3 h and &ggr;-energy of 159 keV, the radionuclide I-123 may be well-suited for medical imaging for diagnostic purposes; its manufacture, however, is costly.
The best choice for diagnostics is Tc-99m which combines a short half-life of 6 h and a radiation energy of 140 keV that is favourable for in-vivo imaging with ready availability as it can easily and cost-effectively be gained in the form of pertechnetate by means of a molybdenum generator and is available, following reduction, at an oxidation number suited for complexing organ-specific substances.
Metals can be complexed directly or indirectly with organ-specific substances.
With direct complexing, functional groups that are contained per se in the organ-specific compound or must be represented by reduction function as ligands for Tc-99m (Schwarz et al., J. Nucl. Med., 28, 721, 1987). Antibodies may thus complex metals following reduction of the disulfide bridges but the need for reduction entails multiple disadvantages. Toxic substances such as mercaptoethanol or dithiotreitol are used as reductants and have to be separated by expensive cleaning processes after the reduction. Quite frequently, the antibody is proved to be fragmented due to the reduction, which can cause loss of antibody affinity for the target tissue in the same way as structural changes due to metal complexing can. In addition, no statement can be made about the exact location of the metal bonding position so that there can be no variation of the groups involved in complex formation, e.g. with a view to optimizing in-vivo stability.
The indirect complexing method uses bifunctional chelates such as DTPA derivatives which, following activation, can either be coupled covalently with organ-specific carriers such as antibodies, or complexed with metals (Meares, Nucl. Med. Biol., 13, 311-318, 1986).
There are basically two ways for conjugate complexing. First, the organ-specific substance is primarily coupled with the free ligand and then complexed with the radionuclide, second, the organ-specific substance is coupled with the chelate complex already formed.
Chemical coupling of the bifunctional chelates with an organ-specific substance requires in both cases sophisticated protective envelope technology as well as cleaning of the complexed conjugate.
Furthermore, protein derivation of the organ-specific substance with a bifunctional chelating agent may impair the specific bonding to the target tissue due to molecular interaction.
In-vivo localization of tumours using isotope-labelled murine antibodies against tumour-related antigens has various disadvantages. Molecular weight is a parameter for biodistribution. There is an inhomogeneous distribution combined with slow blood clearance due to high molecular weight (150 kDalton), in particular, with solid tumours. The result is high background activity which impedes optimum visualization and, together with a high concentration of antibodies in the liver, makes tumour localization difficult if not impossible (Baum et al, Nucl. Med. Commun., 10, 345-352, 1989).
Moreover, many patients show an allergic reaction by forming human antibodies against murine antibodies (HAMA reaction), which prohibits repeated use of said antibodies for diagnostic or therapeutical purposes (Sears et al., J. Biol. Resp. Modifiers, 3, 138-150, 1984). This allergic reaction is attributed to the constant antibody regions: any development to reduce the HAMA reaction is the production of chimeric antibodies having a variable antigen-detecting region of murine origin and a constant region of human origin (LoBuglio et al., Proc. Natl. Acid. Sci. USA, 86, 4220-4224, 1989).
Molecular weight can be reduced by forming antibody fragments such as F(ab)
2
(100 kDalton) and F(ab) (50 kDalton) to retain affinity and increase blood clearance speed (Andrew et al., Eur. J. Nucl. Med., 12, 168-172, 1986), the antibody fragments showing the disadvantages mentioned above as regards isotope labelling. Further reduction of the molecular weight is achieved by visualizing “single chain fragments” (sFv). “Single-chain fragments” have a molecular weight of 27 kDalton and consist of the variable region of the light antibody chain that is coupled through a linker with the variable region of the heavy antibody chain (Bird et al., Science, 242, 423-426, 1988).
Milenic et al. (Cancer Res., 51, 6363-6371, 1991) gave a comparative description of affinity, specificity and biodistribution of a iodinated monoclonal antibody against the tumour-related antigen TAG-72 and the respective F(ab)
2
, F(ab) and sFv fragments. The sFv fragment shows pharmacokinetics suited for diagnostic imaging due to its lower molecular weight. The lower affinity of the monovalent sFv fragment if compared with the complete antibody is thus confronted with its much faster clearance from the blood or body which reduces background act
Conrad Jurgen
Dinkelborg Ludger
Erber Sebastian
Frommel Cornelius
Hohne Wolfgang
Institut Fue Diagnostikforschung GmbH
Scheiner Toni R.
Webb Ziesenheim & Logsdon Orkin & Hanson, P.C.
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