1. Field of the Invention
The invention concerns a diagnosis substance for application in a method for diagnosis of pathological tissue and a method for production of such a diagnosis substance.
2. Description of the Prior Art
In the diagnosis of complex illnesses, differentiation between body tissues with different illnesses, and differentiation between different stages of a pathological tissue, are of decisive importance for the determining an optimal procedure for therapy. It is known to supply a diagnosis substance containing biomarkers to the tissue in question (usually via the blood stream) to detect a specific tissue type. The biomarkers contain a coupling molecule that specifically binds to a molecule (which here is designated as a target molecule) typical of a specific pathological tissue or a specific stage of such a tissue. Binding events at the tissue can be recognized by means of a label (for example a fluorescing dye molecule) detectable with a detection device and linked with the coupling molecule. A “target molecule” means not only individual molecules but also molecular structures that, for instance, have binding locations for a coupling molecule, that are formed from multiple molecules. In addition to biomarkers, the diagnosis substance can contain further components, for instance an aqueous solvent and additives. For example, in the case of the early hgPIN stage (high grade prostatic intraepithelial neoplasia) of prostate cancer, CEACAM-1 molecules (carcinoembryonic antigen-related cell adhesion molecules) are formed in the endothelium of the blood vessels directly adjacent to the cancer tissue. These molecules are thus indicators for the cited cancer stage. The goal of the diagnostics is to reliably establish the presence of such “indicator molecules” or target molecules. A biomarker with which this is possible must tether to the target molecule or the target structure with high selectivity and specificity, meaning that it must be able to “recognize” a specific binding partner from the entire set of possible binding partners with high probability and to reliably bond thereto. Moreover, the biomarker must remain bound at least until the aforementioned detection has concluded. With regard to the detection, it must be ensured that this can be implemented with an (optimally non-invasive) appropriate method.
Complex pathologies such as cancer, among other things, exhibit many characteristics, degrees of maturity and aggressiveness grades in addition to the stages mentioned above. For example, a separate molecular mechanism exists for the metastatic activity of each type of primary tumor (prostate, lung, intestine etc.) as well as given primary tumors for each target tissue of a metastasis. A large number of different biomarkers is required in order to be able to make a differential diagnosis in this context, which is in turn necessary for the selection of an optimal therapy. Methods similar to as in active substance development (drug discovery) are employed for their production. Such a method is, for example, “high throughput screening”, in which many thousands of samples of targets (thus of target molecules) are brought together with thousands of different molecule types in an automated manner. Molecules that bind well are thereby considered as potential biomarkers, but these must then still be optimized with regard to their binding properties, for example in different environmental conditions, as well as with regard to their biocompatibility and their ability to bind to a detectable label. The production of biomarkers according to this method is therefore relatively laborious and complicated.
An object of the present invention is to provide a diagnosis substance that can be produced more simply, as well as to provide a corresponding production method.
The first cited object is achieved by a diagnosis substance according to the invention that contains at least one virus population with specific virus particles binding to target molecules typical of a specific pathological tissue, with a label that is detectable with the use of a detection device being bound to the virus particles. Naturally, only those viruses that are harmless to humans are used. Virus particles or virions have a coating composed mostly of different proteins, these proteins being genetically, variably encoded in the DNA, meaning that the virus can mutate the structure of the proteins, for instance to adapt to different host cells. Due to the genetic variability of the virus coating, the principle of reproduction and selection (thus a directed biological evolution) can be utilized in order to reconfigure a protein of the virus coating so that it specifically binds to the target molecule. M13 phages (completely harmless to humans) that proliferate with E. coli as host cells are particularly advantageous. These are thread-shaped phages with a thickness of approximately 6 nm and a length of up to 1 μm. They contain a single DNA ring that is surrounded by a casing made of a coat protein (here designated with Type 1). Additional proteins that are designated here with Type 2 and Type 3 are located at the ends of the phage. The cited proteins can easily be adapted to the most different binding partners via directed evolution, as is explained in detail further below.
After a diagnosis substance has been supplied (for instance via oral, intravenous or rectal administration) to a pathological tissue (referred to in the following as a prostate cancer) via the bloodstream, a specific binding occurs between a target molecule and a coat protein of a virus particle serving as a biomarker which, in a particularly preferred embodiment variant, is an M13 phage 1 that is exemplarily referenced in the following. M13 phages are a widespread tool in genetics, such that their use is advantageous insofar as known and reliable techniques can be used (for example in their reproduction). The thread-shaped M13 phage 1 has an essentially cylindrical coating extending nearly over its entire length, made from a single type of protein surrounding a single-stranded DNA ring (not shown). Additional proteins of Types 2 and 3 (respectively indicated in
Depending on the pathology in question, different indicator molecules or target molecules 3 act as binding partners for the phages 1. Given the treatment of prostate cancer, in which the present invention is particularly advantageously applicable, different molecule types form in the endothelium of the vascular wall 6 of blood vessels 2 of the cancer tissue 4 or the blood vessels 2 directly adjacent to the cancer tissue for respective different stages of the prostate cancer; for example, molecules known as CEACAM-1 molecules form in the case of the hgPIN stage, thus high-grade prostate neoplasy. In this case the phage 1 is bred (cultured) by the method explained in detail further below so that its coat protein selectively and specifically binds to CEACAM-1. A later cancer stage already located in the angiogenesis stage can be established using the growth factor VEGF or using the alpha(V)-beta(3)-integrins that are likewise formed, for example. The phages 1 used for differential diagnosis of the cited cancer stage are likewise adapted via a directed evolution method so that they selectively and specifically bind to the cited molecules. An administered diagnosis substance can now contain not only one type of a specific phage 1 but rather multiple different phages 1 binding to different target molecules. For example, it is thus advantageous when the diagnosis substance contains both phages 1 binding to CEACAM-1 and phages 1 binding to VEGF and/or alpha(V)-beta(3)-integrins.
The method to produce a specific phage 1 binding to a specific target molecule proceeds as follows (
Although further modifications and changes may be suggested by those skilled in the art, it is the intention of the inventors to embody within the patent warranted hereon all changes and modifications as reasonably and properly come within the scope of their contribution to the art.
Number | Date | Country | Kind |
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10 2007 041 835.5 | Sep 2007 | DE | national |