Patent Application
20090305308
The invention relates to a method for identifying cell-specific protein combination patterns of cell surfaces of circulating cells of the immune system of a human or animal for the identification of different stages of organ-specific chronic inflammatory diseases or tumour diseases. The invention moreover relates to compositions and kits comprising antibodies and/or ligands which can be employed for this method.
After having sequenced the human genome one of the biggest challenges in human biotechnology is to decipher the networks of the proteins in the cell. To address a larger number of proteins directly in a biological structure several methods have been reported, however the number of simultaneously detectable molecular components in one cell is still too low to analyse the spatial architecture of molecular networks.
Methods for identifying cell-specific protein combination patterns is known from U.S. Pat. No. 6,150,173, DE 197 09 348, EP 0 810 428, DE 100 43 470 and WO 02/21137. The known methods are named MELK (Multi-Epitope-Ligand-Kartographie) and are based on earlier observations indicating that at least 17 different tissue antigens can be labelled by using iterative rounds of protein-tagging steps in one and the same tissue section. The MELK technology has the potential to label hundreds of different molecular components of the cell simultaneously unraveling the modes and rules of molecular network architectures.
Assuming that within, and on the surface, of cells proteins are assembled as networks generating the myriads of cell functionalities, it is to be assumed that these proteins are not stochastically distributed, but timely and spatially highly organized: as in the written language, in which letters, following syntactic rules, are assembled to words, the proteins assembled as networks are likely to be topologically determined in every cell. The cell itself is the corresponding protein pattern formation apparatus, or, protein co-compartmentalization machine. Each protein must be at the right time at the right location at the right concentration in a cell to interact with other proteins. Every protein network is characterized by a unique contextual protein pattern. Hence this indicates that every cellular functionality is likely to be detectable as a specific pattern in situ. The entirety of all protein networks in the cell is termed a toponome (topos=place/position; nomos=law). To identify protein networks in situ an appropriate microscopic “reading” technology must fulfil the following conditions: It must be capable (i) to colocalize a large number of proteins (or other molecular cell components) independently in one and the same cell in one experiment; (ii) to visualize the resulting patterns specifically designating a given cell type, or given cellular function (or dysfunction) in situ; (iii) to “understand” the meaning of these patterns by comparing a given detected pattern with a memory store containing the rules for protein assemblies in the cell (toponome dictionary). MELK provides the principle of colocalising extremely large numbers of different molecular components.
EP 0 810 428 B1 also discloses an apparatus consisting of a multi pipette handling unit, a fluorescence microscope, and a CCD camera, apply large tag-libraries (antibodies, lectins or any other kinds of ligands) on fixed cells or tissues to localize their molecular components. Each tag is conjugated to the same specific dye, for example FITC, and applied sequentially on the sample. Each labelling step is followed by an imaging and a bleaching step, resulting in iterative rounds of labelling, imaging and bleaching (repetitive incubation-imaging-bleaching cycles). The result of these imaging procedures is a protein fingerprint (or molecular component fingerprint) of every subcellular ‘data point’ in the corresponding sample. The resulting context-dependent protein information, which would be lost in cell homogenates or lists of proteins, supports the view that protein networks are spatially determined functional units in every single cell (different functions=different cellular protein networks=unique cellular protein patterns). The scheme in
MELK supports the search for relevant patterns by providing large data spaces or ‘reading frames’ to map functional clusters of proteins, i.e. in diseases. From a pure mathematical point of view the gain of information provided by MELK compared to methods extracting proteins from cell homogenates is enormous: given 20 proteins, which are isolated from a cell- or tissue homogenate (classical proteome analysis), and given, that every protein can be detected at 250 different concentrations (0 to 250), the resulting protein profile would contain a maximum number of 25020 different possibilities of protein combinations at different concentrations. However, every cell has the potential to combine the proteins in every single subcellular compartment in a different way. Given, that in every cell out of 1 million cells 2000 subcellular ‘data points’ can be measured (MELK), the resulting maximum of possible combinations would be 1 Mio.×2000×25020. Hence, the biological information increases exponentially using MELK compared to the classical procedures of protein analysis (walking through large proteome fractions with subcellular resolution). Cellular fingerprints can be traced out surprisingly fast, sometimes within hours or few days (6).
WO 01/36939 to which reference is made herewith discloses a mathematical method to define an assembly of proteins exerting a given cell function as protein network motif (formally=combinatorial molecular pattern motif; CMP motif). Thereby every protein is expressed as present or absent related to a certain grey value threshold, determined in each individual MELK data set by an automated approach (present=1, absent=0, 1 bit). Hence, the primary image information consisting in grey value distributions for every protein can be replaced by geometric descriptions of combinatorial binary vectors in x/y/z coordinates (combinatorial molecular patterns, CMPs,
Functional considerations imply that proteins involved in cell-to-cell or cell-to-matrix interactions as well as signal transduction are organized as multimolecular domains on the cell surface. Following the theory of the toponome, the number of existing cell surface proteins contributing to these functions, is likely to be much lower than the number of cell surface selectivities that cells have to establish in a multicellular organism. Hence, a cell must establish the principle of differential combinatorics of these proteins to express the myriads of different cell surface selectivities.
Beside these advantages and benefits of the known methods and apparatus for identifying cell-specific protein combination patterns of cell surfaces there is still the need for a reliable and relatively uncomplicated identification of different stages of organ-specific chronic inflammatory diseases or tumour diseases, especially in the subclinical stages of the diseases.
The invention therefore is based on the object of providing an option for the reliable and relatively uncomplicated identification of different stages of organ-specific chronic inflammatory diseases or tumour diseases and an effective means for the treatment of such diseases and especially of subclinical stages of the diseases.
This object according to the invention is solved by way of a method, compositions, kits as well as applications with the features of the independent claims.
Further advantageous variations of the invention are defined in the subclaims.
The solution of the object is based on the knowledge that high selectivities of cell-to-cell interaction play an important role in the circulating cells of the immune system. These cells, especially lymphocytes, are dedicated to immuno-surveillance. During these physiological processes, but also during chronic inflammatory diseases, these cells leave the blood circulation and selectively enter organs by a specific interaction of their cell surfaces with the cell surfaces of the organ-specific endothelial cells. The inventive method for identifying cell-specific protein combination patterns of cell surfaces of circulating cells of the immune system of a human or animal for the identification of different stages of organ-specific chronic inflammatory diseases or tumour diseases comprises therefore the following steps: a) providing a sample of circulating cells of the immune system of a human or animal showing an organ-specific chronic inflammatory disease or tumour disease and providing a sample of circulating cells of the same cell type of the immune system of a healthy human or animal; b) determining cell-specific protein combination patterns of the cell surfaces of said healthy and pathologically modified cells; c) comparing the resulting protein combination patterns of the cell surfaces of said healthy and pathologically modified cells and subtracting the coincident parts of the protein combination patterns of said healthy and pathologically modified cells, thereby determining a cell-specific protein combination patterns of the cell surfaces of said pathologically modified cells resulting therefrom and d) identifying and characterizing the resulting cell-specific protein combination pattern of the cell surfaces of said pathologically modified circulating cells of the immune system in terms of molecules and its spatial arrangement within the cell as disease specific. The inventive method demonstrates that there is a highly selective recognition and invasion of different organs, generally termed homing, which is specifically encoded in the cell surface toponome encompassing the whole homing grammar. This includes pathological invasion processes, for example in organ-specific chronic inflammatory diseases or tumour diseases. Consequently a complete cell surface toponome fingerprint of circulating immune cells in the blood will contain all relevant homing and invasion addresses. Moreover in organ-specific diseases, it is possible to provide direct images of abnormal invasion addresses. This implies that the method according to the invention is able to classify the cell surface toponome of circulating immune cells and provides highly predictive data sets for the differentiation of diseases and to find organ-specific fingerprints already in the subclinical stages of diseases (
The spatial arrangement of the cell surface proteins of circulating cells of the immune system may be captured in an advantageous variation of the invention via the sequence of the following method steps: a) applying a solution to the cell sample; b) allowing the solution to take effect and removing the solution; c) recording an image prior to or after removing the solution, wherein the solution contains at least one labeled antibody and/or ligand binding specifically to one of the cell surface proteins. This sequence is the core of the already mentioned MELK method which is described in detail in the printed patent specifications U.S. Pat. No. 6,150,173, DE 197 09 348, EP 0 810 428 and to which reference is made herewith. Steps a) to c) may also be repeated with at least one further solution which equally contains at least one labeled antibody and/or ligand binding specifically to one of the further cell surface proteins, and in that possibly subsequent to step b) and/or c) a washing step and/or subsequent to step (c) a bleaching step is performed. The bleaching step may prove necessary or at least advantageous when employing antibodies and/or ligands coupled to the fluorescent dye.
Particularly preferred for the identification of the cell-specific protein combination patterns of the cell surfaces is the employment of at least one antibody which is member of the following group:
Particularly preferred antibodies are fluorescein-labeled or quantum dot labeled antibodies. Besides the fluorescein labelings of course other labelings are possible, too. The named antibodies have proven particularly suitable in a number of tests.
In a further advantageous variation of the method according to the invention the spatial arrangement of the cell surface proteins HLA-DQ, Fcgamma RIII (CD16) and CD11 in the cell sample is captured for identifying an amyotrophic lateral sclerosis (ALS)-specific combinatorial molecular pattern of the cell surface, “HLA-DQ off/Fcgamma RIII (CD16) on/CD11 off”.
In a particularly preferred variation of the invention the MELK robot technology is used for the detection of the disease-specific protein combination pattern of the cell surfaces of said pathologically modified circulating cells of the immune system.
The above mentioned object is also solved by providing compositions or kits, which comprise at least one antibody and/or at least one ligand, wherein the antibodies and/or ligands bind the cell surface protein CD4, CD8, HLA-DQ, HLA-DR, CD3, CD26, CD38, CD45, Fcgamma RIII (CD16), CD57, CD56, CD7, CD71, CD11, CD36, CD19, or CD2 specifically and are coupled each with in particular different labelings. If different labelings are used, the individual antibodies and/or ligands and thus also the cell surface protein bound through them can be differentiated in the case of a simultaneously performed labeling. By employing the composition according to the invention the time required for the identification of the CMP motif is considerably shortened and the performance of the above method facilitated. In a kit the individual antibodies and/or ligands are contained in different receptacles, but are still made available to the user simultaneously. When using such a kit it is not necessary that the labelings of the cell surface proteins are performed simultaneously, so that the antibodies and/or ligands may also be coupled with identical labelings. In a preferred variation of the invention at least one of the labelings is a fluorescent dye, in particular phycoerythrin or fluorescein, or a quantum dot.
The described compositions or kits are suitable for being employed in the identification of different stages of organ-specific chronic inflammatory diseases or tumour diseases and especially of subclinical stages of the diseases as part of a laboratory test.
According to the invention also a biochip is provided, wherein on one surface of the chip ligands and/or antibodies binding specifically to the disease-specific protein combination pattern of the cell surface of pathologically modified circulating cells of the immune system are coupled in such a way that their bondability to the specific protein combination pattern of the cell surface is sustained.
The above-mentioned object according to the invention is also solved by the use of the described inventive composition or kit or biochip for preparing a means of the diagnosis of different stages of organ-specific chronic inflammatory diseases or tumour diseases.
The above-mentioned object according to the invention is also solved by the use of the described inventive composition or kit or biochip for preparing a means of the diagnosis of different stages of ALS. This is because it could be shown that Fcgamma RIII (CD16) plays a central role during the different stages of ALS, so that this process is prevented by inhibiting the Fcgamma RIII (CD16) by aid of a corresponding human antibody.
The above-mentioned object according to the invention is also solved by the use of cell-specific a protein combination pattern of the cell surfaces of pathologically modified circulating cells of the immune system for the development of antibodies directed against this pattern for preparing a remedy for the treatment of organ-specific chronic inflammatory diseases or tumour diseases.
Additional features and advantages of the invention derive from the following description of an example supporting the inventive concept.
The inventive method was applied to the analysis of the cell surface toponome of peripheral blood mononuclear cells (PBMC) using an antibody library against 18 cell surface proteins, most of which belong to the class of adhesion molecules. The antibody library used was directed against the following cell surface proteins (following the cluster of differentiation-antigen-nomenclature):
CD4, CD8, HLA-DQ, HLA-DR, CD3, CD26, CD38, CD45, Fcgamma RIII (CD16), CD57, CD56, CD7, CD71, CD11, CD36, CD19, and CD2.
Briefly, the PBMC were isolated form the blood by a Ficoll gradient, then diluted in PBS, placed on cover slips, air dried, fixed for 10 sec in acetone, and then snap frozen in isopentane pre-cooled in liquid nitrogen. Immediately before use for the toponome measurements according to the invention the cells on the cover slips were fixed in acetone at minus 20° C., air dried and then rehydrated in PBS. The cells were then subjected to measurements as described in the inventive methods described above.
Seven individuals suffering from the sporadic form of ALS and eight healthy control individuals were investigated. Since the methods according to the invention are preferably only performed in the laboratory, the sample-taking preferably is not part of the method according to the invention.
As a major result a statistically significant cell surface toponome motif was found in the ALS patients that was either not observed or with only a very low frequency in normal controls.
The motif carries the following characteristic features:
Together this motif shows that in the blood of ALS patients there is an accumulation of PBMC which are characterized by constant presence of the Fcgamma RIII receptor (1), which, by a rule, is anti-colocalized for CD11 (0) and HLA-DQ (0), while the other cell surface proteins indicated as wild cards (*) are variably associated with Fcgamma RIII, e.g. may be present (1)(on) or absent (0)(off) in may different combinatorial variations in individual single PBMC (characteristics of a motif by definition). Together 246 variations (cell types) of this motif were found. PMBC carrying this cell surface toponome motif are statistically significant in ALS (p=0.019, T test).
The sporadic form of ALS is characterized by cell death of motor neurons in the CNS (pyramidal tract), and also involves the second peripheral motor neuron, whose cell bodies are located in the anterior horn of the spinal cord. While the cause of this neuronal cell death is unknown, it is well known that ALS is accompanied by presence of some CD8 positive immune cells in the spinal cord pyramidal tract, which represent the myelinated axons of the first central motor neuron projecting to the anterior horn cells of the spinal cord to link the central with the peripheral motor neuron.
Upon routine current classic immunocytochemical techniques these CD8 positive cells are not conspicuous to directly alter the motor neuron axons (McGeer P L M & McGeer E G: Muscle Nerve 26, 459-470, 2002). Therefore the current scientific opinion is that these cells invade the spinal cord as a secondary response to the degeneration of the first motor neuron, rather than playing a primary part in motor neuron death mechanisms.
In order to investigate this issue by using the inventive method, post mortem spinal cord tissue was analyzed using the same above described antibody library.
The following post mortem tissues provided were studied:
As a result of these studies the following new features of ALS were found:
In the lumen of post capillary venules which are present in the pyramidal tract of the spinal cord, single PBMC were found during their penetration of the capillary endothelium (a process that is commonly defined as homing).
These same cells expressed precisely the abnormal cell surface toponome phenotype as the PMBC found to be specifically occurring in the blood of ALS patients: The same toponome motif, with Fcgamma RIII as a lead protein (see above).
Several different stages of this homing process were found showing that the Fcgamma RIII is internalized at the rear polarized site in these cells while the front site protrudes across the endothelium. After this penetration process the cells are located between the myelinated neurons projecting thin long cell extensions penetrating the myelin sheath of morphologically intact single motor neurons forming a hook-like anchor within the motor axon. In that stage of penetration the cells do not show any expression of the Fcgamma RIII but a strong co-expression of CD3, CD8 and HLADR at the very tip indenting the axon.
By analyzing the normal control spinal cords, homing phenomena of single PBMC were also seen, but by striking contrast, these cells never expressed the Fcgamma RIII, and instead of CD8 co-expressed CD4, while the other associated molecules were different from those seen as a motif in ALS (absence of the specific variations of protein combinations present in ALS). Another striking difference of these normal homing phenomena was that after penetration of these cells to the motor neurons, these cells never formed cell projections, but instead reside strictly between the myelin sheaths without attacking the motor neuron myelin.
There are several new conclusions based on the inventive method that now can be drawn concerning the cellular pathogenesis of ALS.
1. The peripheral ALS-Toponome motif on blood PBMC is obviously a predictive combinatorial molecular phenotype of ALS, because PBMC with precisely the same cell surface toponome motif home to the spinal cord pyramidal tract. In contrast to current knowledge it is now clear for the first time that ALS pathogenesis relies on specific abnormal immune cells detectable in the blood.
2. The fact that these invasive PBMC axotomize motor neuron axons by penetrating the myelin sheaths strongly suggests that these cells actively and selectively damage the myelinated axons of the first motor neuron. While the cause of this specific attack is not known at present, this result provides direct evidence that the detected abnormal PBMC type plays an active role in the motor neuron damage. This result relies on the high “functional” (multi-molecular combinatorial) resolution of the inventive method thereby unraveling a more active role of invasive PBMC than previously thought.
These data strongly support the new concept of the generative grammar of the cell surface toponome to produce a large number of highly selective invasion addresses by differential combination of cell surface proteins generated by PBMC.
Most importantly, by applying the method according to the invention to PBMC it is now possible to progressively decipher the combinatorial invasion/homing code of PBMC in the blood in many diseases, such as chronic inflammatory diseases, e.g. cellular autoimmune diseases (chronic hepatitis, diseases of the rheumatoid class, thyroiditis, Multiple sclerosis) and in cancer, in which also selective PBMC invasion/homing into the tumour sites is observed. Hence analyses according to the inventive method of blood samples in these diseases will provide specific cellular toponome fingerprints predictive for both the diseased organ, and most likely the type of disease. The above described antibody library is claimed to be of central importance for such detection and prediction procedures based on the present inventive analyses of the blood.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2006/002569 | 3/21/2006 | WO | 00 | 10/27/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60663834 | Mar 2005 | US |
