FR2894032A1 - Method for detecting tumors and assessing their sensitivity to chemotherapy, by detecting or quantifying specific proteins or antibodies, also treatment using interfering RNA against these proteins - Google Patents

Abstract

Method for detecting (a) presence/absence of a tumor in a mammal and/or (b) its sensitivity to chemotherapy comprises detecting and/or quantifying, in a sample from the mammal, (i) eEF1a1 protein (I; elongation factor of protein synthesis); (ii) antibodies (Ab1) against (I) or a fragment of it containing at least one epitope; (iii) MARK3 protein (II) and/or (iv) antibodies (Ab2) against (II) or a fragment of it containing at least one epitope. Independent claims are included for the following: (1) diagnostic kit for the new method; (2) Ab1 and Ab2 as new compounds; (3) method for inhibiting growth of tumor cells in vitroby inhibiting activity of (I) and/or (II) using either Ab1/Ab2 or an interfering RNA (iRNA) that inhibits expression of the genes encoding (I) and/or (II); (4) iRNA that inhibits, in vitroor in vivo, expression of genes encoding (I) and/or (II); (5) vector for expressing the iRNA of (4) containing the sequence encoding iRNA under control of regulatory elements; (6) vector for delivering iRNA in a host cell comprising iRNA of (4) and a system for delivering it; (7) a specific variant (V1) of MARK3; (8) nucleic acid sequence that encodes V1; (9) expression vector containing the sequence of (8) and regulatory elements; (10) host organism containing the vector of (9); and (11) method for producing V1 by growing the host of (10). ACTIVITY : Cytostatic. MECHANISM OF ACTION : RNA interference; antibody neutralization of protein activity. U373 glioblastoma cells were transfected with the siRNA 5'-acagcacuauuccugauca 5'-ugaucaggaauagugcug directed against MARK3. After culture for 5 days, the treated cells had a level of proliferation 74% of that in untreated controls.

Description

Method for demonstrating the presence or absence of

markers associated with tumors

  The present invention relates to a novel method for demonstrating the presence or absence of tumor-associated markers. The invention also relates to diagnostic kits comprising the means for carrying out the method according to the invention as well as the use of compounds that inhibit the activity or the expression of said markers for inhibiting the growth of tumor cells.

  In the field of pathologies, cancers occupy a preponderant place in terms of prevalence, incidence and mortality. The phenomena of cancerization are related to complex cellular disorders, imperfectly known, which can affect all organs. The means of cancer screening and control remain imperfect. There is a wide variety of tumor phenomena that can occur at all ages of an individual and affect most functional areas of a human organism. In particular, the central nervous system (CNS), a complex organ composed of many different cell types, does not escape these morbid and very diverse pathological phenomena. In fact, there are many types of solid brain tumors. These tumors correspond to the development of oncological phenomena affecting the constituent cells of the CNS themselves (neuronal cells, glial cells, etc.). There are also other tumors located in the CNS that are the result of metastases from tumors of other organs. CNS tumors are characterized by a number of anatomical, biological and clinical parameters. At present, the careful analysis of these parameters predominantly conditions the therapeutic action strategy initiated by the clinician. The specific phenotype of tumors is an element, which makes it possible, very imperfectly, to evaluate in a prognostic way the chances of survival of the patient. Among the explorations to classify brain tumors are: - Medical imaging (computed tomography and magnetic resonance imaging) - Morphological and histological analysis performed on biopsies - Biomolecular analysis: search for protein markers by immunoassay detection, cytogenetic analysis (eg, detection of genetic macro-anomalies by hybridization of probes) ... The diagnosis of tumors and more particularly for CNS tumors is based predominantly on a histological analysis performed by the pathologist.

  Unfortunately the diagnostic discrepancies observed between experts of the field are enormous (up to 64% of disagreement according to the tumors). Worse, similar discrepancies can be noted when interpretations of identical samples are given to the same person within a few weeks of each other (Mittler et al, 1996, Bruner et al, 1997, Coons et al, 1997). This is worrying given that misdiagnosis can lead to unnecessary radiotherapy and / or chemotherapy that has serious consequences for the patient. In addition to histological analysis, there are only a few diagnostic tests based on molecular approaches. Cytological observations can thus be supplemented by the search for genetic abnormalities and in some laboratories the detection of certain protein markers using specific antibodies. For the time being, there are no routine tests based on the detection and quantification of the concentration of transcriptomic markers of tumors. The few molecular tests currently available do not make it possible to distinguish unambiguously the different types of tumor cells and above all, to correctly predict their sensitivity to cytotoxic agents. It is therefore important to be able to have new methods for easily, substantially and early detection of the presence of tumors in order to implement the therapeutic strategies best suited to the treatment of each patient. The present invention thus relates to a novel diagnostic method for detecting the presence or absence of a tumor in a mammal, in particular in humans, by detecting and / or quantifying the presence of a new biological marker in a mammal. biological sample previously taken from said mammal: a MARK3 protein. The presence of a MARK3 protein or of antibodies directed against a MARK3 protein or against a fragment comprising at least one epitope of said protein is characteristic of the presence of a tumor. The present invention thus relates to a method that can be used to detect the presence or absence of a tumor in a mammal, said method comprising a step of detecting and / or quantifying on a biological sample previously taken from said mammal: the presence of a MARK3 protein, and / or the presence of antibodies directed against a MARK3 protein or a fragment comprising at least one epitope of this protein. According to the invention, the term "MARK3 protein" is intended to mean a protein which comprises a protein sequence of the reference MARK3 protein, described in GenBank Accession No. AF387637 (Sun T.-Q. et al., "PAR-1 is a Disheveled-associated kinase and a positive regulator of signaling ", Nat Cell Biol., 2001, 3, 628-636), its isoforms, variants and biologically active fragments. The isoforms, fragments or biologically active variants according to the invention are recognized by the anti-MARK3 antibodies. According to the invention, variants are understood to mean proteins which advantageously have at least 75% identity with the reference MARK3 protein, more preferably at least 80%, more preferably at least about 85% identity. The methods of alignment and calculation of sequence identities are well known to those skilled in the art and accessible directly on the Internet. One particular mention is the BLAST program, which can be used from the site http://www.ncbi.nlm.nih.govBLAST/ with the parameters indicated by default on this site. It is also advantageous to use the advanced search of BlastP by refining the search with a pattern (PHI-BLAST). For the alignment of the sequences, one can use the programs CLUSTALW (http://www.ebi.ac.uk/clustalw/) or MULTALIN ((http://prodes.toulouse.inra.fr/multalin/), with the parameters indicated by default on these sites.The differences between the MARK3 variants and the reference sequence may be due to deletions of at least one amino acid and when several amino acids are deleted they may be contiguous or separated on the The differences can also be due to mutations, with at least one amino acid being replaced by a different amino acid in the reference sequence, and the differences can also be due to the addition of at least one amino acid to the amino acid. Within the reference sequence, when several amino acids are added within the reference sequence, they can be contiguous, that is to say forming a protein fragment of at least 2 amino acids, or distributed over the sequence. e or both.

  According to one particular embodiment of the invention, the variant of MARK3 comprises at least one protein fragment inserted into the reference sequence MARK3. This fragment can comprise up to 100 amino acids, generally between 10 and 60 amino acids. According to a particular embodiment of the invention, the MARK3 protein is a variant comprising the protein sequence represented in SEQ ID NO.

  Advantageously, the biological sample previously taken is selected from a sample of blood serum, lymph, cystic fluid, tissue homogenates, preferably a sample of blood serum. It is understood that according to the detection and / or quantification method used, the biological sample taken may be subjected to a prior treatment for its analysis, for example grinding and / or dissolution. Such treatments are well known to those skilled in the art in connection with the detection methods. The means for detecting and / or quantifying the presence of a protein or antibody in a biological sample are well known to those skilled in the art, in particular described hereinafter and in the examples.

  The methods described below are presented for information only and do not constitute in any way an exhaustive list of the technical approaches that can be used to implement the invention. Those skilled in the art will be able to identify, according to the state of evolution and optimization of the techniques, at any time, which are the most suitable methods for carrying out the analyzes intended to implement the invention while advantageously substituting the methods measurements and / or immunoreactions described herein. The following methods are presented for the sake of simplification in the following groups: • Tests involving media capture • Detection tests based on the macroscopic detection of immune complexes • Tests based on isotopic markings and RIA (radio- immunoassays) • Analyzes based on immunohistochemistry techniques • Tests based on fluorescence transfer methods, FRET (Fluorescence Resonance Energy Transfer), or energy transfer based on luminescence BRET (Bioluminescence Resonance) Energy Transfer) Tests involving media capture In the methods described below, mention is made of the use of antigens corresponding to a MARK3 protein or a fragment of this protein comprising at least one epitope recognized by the anti-MARK3 antibodies. It is therefore understood that the tests can also be carried out with fragments of only these protein-antigens (natural or synthetic or chimeric peptides, etc.) or haptene-type compounds and molecules capable of selectively reacting with the anti-antibodies. -MARK3 whose presence in the sera is sought. The Western blot and the ELISA method described hereinafter are two of the most commonly used methods for such tests. Other methods of detecting antigen-antibody reactions can be employed using various modes of interaction or adsorption of the components with supports, reaction wells or microdetection systems. These tests often combine high levels of performance, related to the high sensitivities of the methods, the relative simplicity of sample handling, the robustness of the tests, and the high throughput processing capabilities of many samples simultaneously. a) method based on the Western blot (Towbin et al., 1979; Burnette, 1981) For carrying out this method, an extract containing at least one antigen according to the invention is subjected to electrophoretic migration in polyacrylamide gel in medium denaturant. This technique is well known in one of these variants under the acronym of SPAGE: for "SDS 25 Polyacrylamide Gel Electrophoresis"; or polyacrylamide gel electrophoresis in the presence of the denaturing detergent Sodium Dodecyl Sulfate. Electrophoretic migration allows proteins to be separated according to their respective molecular weights (Laemmli, 1970). At the end of this separation, and according to standard protocols known to those skilled in the art, fingerprints of the proteins are made on a nylon membrane and are incubated with the sera to be analyzed. Western-blot detection protocols, very conventional, make it possible to reveal the binding of the antibodies, which were possibly present in the sera, on the antigens fixed on the fingerprint. The protocols make it possible to reveal by radioactive, fluorescent or luminescent techniques the presence of the antibodies. The analysis can be conducted in such a way as to access a sufficiently accurate quantitative estimate of the level of antibody in the starting sample to make a diagnosis of clinical interest. This kind of test can be performed using the equipment necessary for producing Western blots distributed by Immunetics (Boston, Mass., USA; http://www.immunetics.com). b) ELISA Method (and its Derivatives) One of the now-standard methods, called Enzyme Linked Immuno Sorbent Assays (ELISA) (Engvall and Perlmann, 1971, 1972, Engvall et al, 1971) consists in causing the creation of antigen-antibody complexes ( complexes-immune) in immobilized form on the walls of the wells (wells) of plastic multi-well dosing plates. This type of method is available in a multitude of variants depending on whether the protocols are based on the initial immobilization of antibodies or antigens in the bottom of the wells and according to the revelation methods used (direct or indirect ELISA). By way of example, it may briefly be mentioned that this kind of test can be performed according to the description which follows for the detection of the antibodies directed against the antigen according to the invention. Thus, the wells of the ELISA assay plate are individually and independently filled with increasing dilutions of the antigen. The proteins are adsorbed on the bottom of the wells. After washing the wells, the adherent proteins on the walls of the wells are contacted with the desired antibodies and present in the sera. Therefore, if present in the sera, the antibodies will immobilize in the bottom of the well by attachment to the antigen proteins adsorbed on the wall of the well. A detection step (based for example on a simple colorimetric test) of the presence of antibodies at the bottom of the wells indicates consequently the presence of these antibodies in the starting serum sample. Conducting assays on sample dilutions provides a quantitative estimate of antibody levels in the biological sample. Analyzes of ELISA tests can be performed on devices such as the VIDAS system distributed by Bio-Mérieux (http://www.biomerieux.com). c) methods based on protein microarrays In an immobilization approach of immune complexes, the micro-arrays technique is based on a logic of miniaturization, automation and more or less massive parallelization of the number of tests. For these tests, it is necessary to create micro-arrays of proteins consisting of generally flat solid surfaces (glass slides, silicon fragments, wells of multi-well plates of plastic material, etc.) comprising antigens fixed by various chemical processes on the support, each antigen being deposited on a small surface of the support representing a few micron-squares of surface. For example, the antigens are immobilized on the support by deposition of antigen suspension microdroplets on these supports (Peluso et al, 2003, Kusnezow and Hoheisel, 2003). After incubation with the samples to be analyzed, the formed immune complexes can be detected by various technical means, the most common being based on the detection of fluorescence signals or by a method using surface plasmon resonance (Vikinge et al., 1998). Kusnezow and Hoheisel, 2003).

  Fluorescence-based microarray-based assays can be envisioned according to protocols that even permit analysis directly on a whole blood sample. The system, such as that proposed by Umedik (http://www.unedik.com/) comprising micro-array and reader allows the analysis of several markers simultaneously with quantization signal intensities. Surface plasmon resonance is based on a well-known experimental physics principle where the flat surface of a gold film reflects an incident light ray in a direction predicted by the laws of classical optics. However, for a very small part of the reflected light beam and at a certain incidence, there is a significant decrease in the number of reflected photons. The angle of incidence of the zone of reflection of the less luminous zone depends on the quantity of material fixed on the face of the gold film opposite to the face irradiated by the incident light beam. Any interaction of antibodies, antigens or complex-immune complexes on the opposite face of a detector based on plasmon resonance and irradiated at a certain incidence by a light ray thus causes a significant change in the angle of reflection of the less luminous part of the reflected light beam. The detection of this deflection of the reflection angle makes it possible to highlight and quantify the fixing of components on the detector.

  This last detection method is at the basis of a technique for analyzing the interactions between antibodies and proteins and makes it possible to measure the interaction kinetics parameters between antigens and antibodies and to deduce therefrom the quantities of antibodies present in a molecule. sample (Fagerstam et al, 1990, Szabo et al, 1995). This technique is developed in the form of measurement automata, an example of which is known as BlAcore (BlAcore AB, Uppsala, Sweden, http://www.biacore.com). d) Method Based on Mass Spectrometry The detection of the antibodies can be carried out using, for example, so-called SELDI ™ Surface Enhanced Laser Desorption / Ionization (TFT) technology (Merchant and Weinberger, 2000; al, 2000). This embodiment is carried out by exploiting the technical platform distributed by Ciphergen (Ciphergen Biosystems, Fremont, CA, USA, http://www.ciphergen.com). Detection of the antibodies can be achieved by pre-immobilizing the antigens on the affinity supports usable on the Ciphergen mass spectrometer or any other mass spectrometer suitable for this kind of analysis. It is indeed possible to immobilize antigens by chemical grafting on the supports of silica, metals or polymers and to use these supports to trap the antibodies present in a sample to be analyzed. The immune complexes thus formed and immobilized on the supports can then be analyzed by mass spectrometry. In this type of analysis, the peak immunoglobulin bound to the antigens can be detected in the mass spectrum. The use of the grafted antigens on the support makes it possible to constitute an extremely sensitive and specific assay method. Given, on the one hand, the affinity of the antibodies for the antigens, and on the other hand the high detection sensitivity of the mass spectrometer, minute quantities of antibodies present in a sample can be detected.

  Detection tests based on the macroscopic detection of immune complexes (latex test, immuno-detection strips) (Singer et al., 1957, Hechemy et al, 1974) In this type of detection system, the antigens of The interest is chemically coupled to particulate components of micrometric sizes such as colored or unstained polymer beads. Incubation in a liquid or semi-liquid medium of a fluid suspension of these antigen-coated beads with the biological sample to be analyzed leads to the creation of immune complexes that aggregate several polymer beads together. This aggregation results in the formation of bundles of balls whose size becomes macroscopically important to the point of being visible to the eye by an operator. In a common and sophisticated variant of the system, the immune complexes are subjected to capillary migration on a chromatography support strip and revealed by creating a colored band indicating the presence or absence of the detected antigen (symbolized test). immunodetection strips widely used routinely for example for pregnancy tests). Latex tests are marketed by Bio-Mérieux for microbiological diagnostics for example (www.biomerieux.com).

  Tests based on isotopic markings and RIA (radioimmunoassays) (Yalow and Berson, 1960, Booth et al, 1982) In one of the variants of this assay, the immune complex is produced by adding to the reaction medium, in addition to the serum sample, a known amount of radiolabelled isotope labeled antigen. After selection of formed immune complexes, the amount of detectable radioactivity in the fraction thus isolated is proportional to the amount of antibody present in a sample. Diagnostic kits using the principle of RIA are distributed for example, for various dosages, by Schering / Cis-Bio International (Gif / Yvette, France, www.cisbiointernational.fr). "Immunoassay" may also be based on a dosing principle not using radioactive tracers but fluorescent markers or luminescence. Analyzes Based on Immunohistochemistry Techniques (Kiernan, 1999) A robust and relatively simple approach in principle is to perform an immunohistological analysis in sections, smears or other preparations from biopsies. In this case, these are tests performed on a raw sample (a section of tumors made up of more or less homogeneous cells). The application of this technique is therefore more generally intended to highlight the antigens in the preparation. Demonstration of formed immune complexes requires the detection of a signal generated by the use of radioactive tracers, or fluorescent reagents or colorimetric methods. The tumor cells containing the antigens will show a positive reaction with the selected antigen specific reagent. Conversely, non-tumor cells will not show a signal or signal of significantly lower intensity. Tests based on fluorescence-based cell sorting (Fluorescent Activated Cell Sorting (FACS) method (Hulett et al., 1969, Parks and Herzenberg, 1984): a fluorescently labeled reagent may be used to label whole cells derived from and isolated from biopsies and to allow the sorting and quantification of positive cells for the presence of the desired antigens. As part of this method, unlysed cells dissociated from freshly collected biopsic tissue are contacted with the fluorescent selective reagent and the suspension and then analyzed using a cell sorter. The cell sorter individually detects the intensity of the fluorescent signal associated with each cell, and counts the number of cells detected and optionally isolates the cells in specific reservoirs. Instruments designed for FACS analysis are distributed for example by Becton Dickinson (Franklin Lakes, NJ, USA, http://www.bd.com). The use of appropriate and optimized technical parameters (cell dilution, optical parameters, ...) makes it possible to consider sorting and selective counting of the cells containing the antigens and to ensure the feasibility of this method. It should also be noted that a flow cytometry approach applied to the analysis of the sera could be carried out using beads labeled with specific fluorescent compounds and comprising antigens grafted onto their surfaces. The immune complexes can then be demonstrated for example with the Bio-Rad Bio-Plex technology (Hercules, CA, USA; http://www.bio-rad.com); or the one named Becton Dickinson's "Cytometric Bead Array" (http://www.bdeurope.com) or the Miraibio Luminex system (www: //www.miraibio.com).

  Tests based on fluorescence transfer methods, FRET (Fluorescence Resonance Energy Transfer) or BRET (Bioluminescence Resonance EnergyTransfer) For such an analysis, the dosage can, in its most attractive version, be carried out directly in solution (homogeneous phase assay ) and does not require isolating or purifying any of the components of the immune complex. This method requires in one of these variants the use of two different antibodies directed against an antigen and labeled with appropriate fluorescent groups. The two fluorescent groups are selected so that their optical characteristics make it possible for one of the groups to be excitable by the light radiation used for the fluorescence measurement, and then allow the transfer of the excitation energy to the second fluorescent group. which emits, as a last resort, a fluorescence radiation of very specific wavelength. The fluorescence transfer is effective only if the two molecules are kept close enough close to each other. The two antibodies labeled by the two fluorescent groups are chosen to be able to bind simultaneously to the antigens. The ternary immune complex formed (fluorescent antibody-antigen antibody - fluorescent light emission antibody) thus allows a combination of two antibodies and in this case only, a fluorescence signal can be detected (at the wavelength d emission of the fluorescent emission group). In fact, this type of assay is rather suitable, without exception, for the determination of antigens. The intensity of the measured fluorescence signal is therefore directly proportional to the amount of antigen present in the biological extract (Mathis 1995, Szollosi et al., 1998, Blomberg et al., 1999, Ueda et al., 1999, Enomoto et al. , 2000). The protein analysis by the fluorescence transfer method can be carried out on the Kryptor apparatus of the German company B.R.A.H.M.S. (Www.brahms.de).

  Alternatively, the fluorescent compound excited by excitation light radiation in the FRET technique may be substituted by a bioluminescent system that relies on the activity of an enzyme (Xu et al, 1999). The non-exhaustive list of techniques described herein above is an example of the various techniques that a person skilled in the art is able to use to perform an analysis of biological samples in order to implement the invention to detect the antibodies directed against the identified antigens, analysis of which it will be possible to derive information of diagnostic or prognostic value in accordance with the invention. The analyzes may be carried out directly on raw samples or having undergone treatments, and corresponding in a non-exhaustive manner to lysates, extracts or subfractions 15 from these samples. The detection and the determination of the antibodies directed against the antigens, described by the invention, can be carried out by using any products or derivatives resulting from these antigens as well as on their precursors if they comply with the criteria of specific recognition with the antibodies to be detected. As indicated previously, the assay of the antigens themselves or their fragments or metabolic modification products may also constitute an analytical application of clinical interest. Tests based on the use of SELDI-TOF mass spectrometry require affinity media for specific antigen capture (Ciphergen ProteinChip arrays). These supports will be adapted according to the chosen capture mode: supports with chemical reactivity adapted to the attachment of antigens, or monoclonal or polyclonal antibodies recognizing all or part of the antigens or their peptides. As is obvious to those skilled in the art, the methods use for quantification purposes, and for quality-control (control-positive) requirements, to appropriate standard proteins, whether or not related to the antigens. In addition, immunodetection tests require the availability of components (components that we will call reagents) that can interact with the antibodies of interest or the antigens and their derivatives. With regard to these reagents, mention may be made, of course, of polyclonal or monoclonal antibodies as well as their immunoreactive fragments, grafted or not on or with other components; particulate elements capable of interacting with antigens (phages or recombinant bacteria expressing on their surface polypeptide regions capable of interacting with haptens or antigens) (Gao et al, 1999, Knappik et al, 2000); or aptamers (chemical molecules of the polynucleotide or even polypeptide type capable of establishing high affinity non-covalent interactions with target molecules) (Ellington and Szostak 1990, Tuerk and Gold 1990). Specific reagents for the detection of immune complexes formed during the tests may be chosen from conventional detection systems suitable for this kind of immunodetection tests such as Western blot or ELISA (these reagents are for example antibodies secondary enzymes coupled to enzymatic systems allowing the execution of colorimetric reactions). Such reagents are available on catalog, for example on the Sigma Aldrich catalog, available online (http://www.sigmaaldrich.com). According to a first embodiment of the invention, the presence of the MARK3 protein is detected and / or quantified by means of antibodies directed against the MARK3 protein or at least one epitope of this protein. The antibodies are advantageously chosen from polyclonal antibodies or monoclonal antibodies. The methods of identification and the preparation of such antibodies are known to those skilled in the art by employing the usual techniques for the preparation of such antibodies. The methods described in Immunobiology (5th ed., Janeway, Charles A., Travers, Paul, Walport, Mark, Shlomchik, Mark, New York and London: Garland Publishing, c2001) will be noted for reference. According to a second embodiment of the invention, the presence of antibodies directed against a MARK3 protein or against a fragment comprising at least one epitope of this protein is detected and / or quantified by means of an antigen comprising at least one epitope of a MARK3 protein.

  According to a particular embodiment of the invention, the antigen comprises a MARK3 protein as defined above and hereinafter. Of course, the antigen may comprise simple fragments of a MARK3 protein, it being understood that said fragment comprises at least one epitope recognized by the anti-MARK3 antibodies. A fragment comprising at least one epitope of the MARK3 protein is, for example, a fragment encompassing the N-terminal portion of the MARK3 protein, more particularly an 11 kDA fragment encompassing this N-terminal portion. An epitope is the smallest structural unit of an antigen recognized by an antibody, a structure present on the surface of the antigen molecule, capable of combining with a single antibody molecule. From a structural point of view, the epitopes can be of two types: linear epitopes (short sequence of amino acids recognized by an antibody), having a size of about 8-10 amino acids, or conformational epitopes, that is, the antibodies recognize amino acids that are spatially close when the protein has its folded structure but are not localized in the immediate vicinity of the protein sequence. It should be noted that a given epitope can be recognized by several different antibodies generated in the context of distinct immune reactions, linked to different agents (viruses, bacteria, etc.). In this respect, the level of recognition between the epitope and the different antibodies may vary from one epitope-antibody pair to another. Thus, if the epitope is strongly recognized by the antibody, low concentrations of antibodies will be sufficient to detect a recognition reaction between the epitope and the antibody. Conversely, if the epitope is weakly recognized by the antibody, high concentrations thereof will be required to detect a recognition reaction between the epitope and the antibody. In the same way, several distinct epitopes can be recognized by the same antibody. But again, the recognition levels of one epitope-antibody pair to another are generally variable. Means for identifying epitopes, antigenic fragments and for preparing antigens useful in a diagnostic method on biological samples are well known to those skilled in the art. In particular, a method consisting in systematically synthesizing peptides containing overlapping fragments of MARK3 protein will be used to test their ability to stimulate an immune response. Those skilled in the art will therefore be able to identify the epitope or epitopes best suited for carrying out the method according to the invention by simple routine experimentation. Preferably, the method according to the invention comprises an additional step of comparing the results obtained in the detection and / or quantification step with a reference value characteristic of the presence of a tumor and / or with a value of characteristic reference of the absence of tumor. These reference values may be different depending on the means used for the detection and / or quantification of MARK3 or anti-MARK3 antibodies. They can be obtained according to usual methods where the same analyzes will be carried out on samples from healthy individuals on the one hand and from individuals known to be carriers of tumors on the other hand. Of course, the method according to the invention may further comprise the detection and quantification of at least one other biological marker characteristic of the presence and / or invasiveness of a tumor and / or its chemoresensitivity. By way of example, mention may be made of the KI67 antigen protein as a proliferation marker (SwissProt reference: http://www.expasy.org/uniprot/P46013), or the phosphorylated vimentin, the absence of which is characteristic. an invasive tumor (PCT / EP2005 / 054598 filed on September 15, 2005) or antibodies directed against eEF1A1 protein (elongation factor for protein synthesis, Swiss-Prot reference: http://www.expasy.org / uniprot / P68104) whose absence or low concentration is characteristic of a chemoresistant tumor. According to a particular embodiment of the invention, the method also comprises a step of detecting and / or quantifying on a biological sample previously collected: the protein eEF 1A, and / or the antibodies directed against the eEF1A1 protein or at least one epitope of this protein. The detection and / or quantification of the two markers can be performed simultaneously, separately or shifted in time, on the same biological sample or on different samples.

  The present invention also relates to a diagnostic kit for implementing a method as defined above and hereinafter, said kit comprising means for detecting and / or quantifying on a biological sample previously collected: the presence of a MARK3 protein, and / or the presence of antibodies directed against a MARK3 protein or a fragment comprising at least one epitope of this protein. Such means are well known to those skilled in the art, defined above, their shape varying according to the selected detection mode. They comprise on the one hand a previously defined MARK3 antigen or an anti-MARK3 antibody according to the invention and reagents necessary for carrying out the diagnostic method according to the invention. Such reagents are well known to those skilled in the art, depending on the detection / quantification method employed. They are in particular described in the references cited above and in particular in the catalog of Sigma Aldrich, accessible online at 15 (http://www.sigmaaldrich.com). Advantageously, the detection kit comprises a suitable support, able to receive the biological sample and the appropriate detection means. When the detection means is an antibody or an antigen, or fragments thereof, it may be bound to the support by any suitable means, for example a covalent bond or adsorbed on the support. Such supports are well known to those skilled in the art, described in particular in the references mentioned above. The present invention also relates to the antibodies directed against a MARK3 protein or at least one epitope of this protein, which specifically bind to a MARK3 protein or at least one epitope of this protein defined above and hereinafter. The invention also relates to said antibodies for their use in therapy. In addition to the invention of MARK3 as a marker for the presence of tumor cells in mammals, particularly in humans, the inventors also found that inhibition of the activity or expression of MARK3 inhibited the growth of tumor cells. The present invention therefore also relates to a method for inhibiting the growth of tumor cells, characterized in that the expression or activity of the MARK3 protein is inhibited by means of an anti-MARK3 inhibitor. The MARK3 inhibitors inhibiting the activity of MARK3 act on the MARK3 protein preventing or limiting its ability to exert its biological function. It may be for example an antibody whose antigen is a MARK3 protein or at least one epitope of this protein, as defined above and hereinafter. The inhibitors anti-MARK3 inhibiting the expression of MARK3 act at the level of the transcription of the gene coding for MARK3 or at the level of the translation of the RNA towards the protein. In this second case, it may be an interfering RNA that hybridizes to the messenger RNA (mRNA) gene expression product comprising the sequence encoding the MARK3 protein to inhibit the translation, either by simple steric hindrance either to promote the cleavage of the mRNA. Interfering RNA technologies and their use in vitro and in vivo are well known to those skilled in the art, described in numerous scientific articles and other patent applications. Depending on the interfering RNA sequences selected by the skilled person, different levels of inhibition can be obtained, to modulate the desired inhibitory effect. Preferably, the interfering RNAs are prepared and selected to obtain at least 50% inhibition of the expression of the target gene in a cell, or even at least 75%, 90%, 95% or even more than 99% inhibition. . SiRNAs, for Small Interfering RNA or small interfering RNA, are short sequences of about 15 to 30 base pairs (bp), preferably 19 to 25 bp. They comprise a first strand and a complementary strand identical to the targeted region of the RNA of the target gene.

  The design and preparation of siRNAs and their use for in vivo and in vitro cell transfection are well known and widely described in numerous publications, such as: US 6,506,559, US 2003/0056235, WO 99/32619, WO 01/75164, WO 02/44321, US 2002/0086356, WO 00/44895, WO 02/055692, WO 02/055693, WO 03/033700, WO 03/035082, WO 03/035083, WO 03/035868, WO 03/035869, WO 03/035870, WO 03/035876, WO 01/68836, US 2002/0162126, WO 03/020931, WO 03/008573, WO 01/70949, WO 99/49029, US 6573099, WO 2005 / 00320, WO 2004/035615, WO 2004/019973, WO 2004/015107, http://www.atugen.com/sirnatechnology.htm, http://www.alnylam.com/science-technology/index.asp, http : //www.protocol-online.org/prot/Research Tools / Online_Tools / SiRNA Design /, http://www.hgmp.mrc.ac.uk/Software/EMBOSS/Apps/sirna.html, http: // www.rockefeller.edu/labheads/tuschl/sirna.html, http://www.upstate.com/browse/categories/siRNA.q. SiRNAs can be designed and prepared using appropriate software available online, for example: - "siSearch Program" http://sonnhammer.cgb.ki.se/siSearch/siSearch_1.6.html ("Improved and automated prediction of effective siRNA ", Chalk AM, Wahlesdelt C, and Sonnhammer ELL, Biochemical and Biophysical Research Communications, 2004). "SiDirect" http: //design.rnai.ip/sidirect/index.php (Direct: highly effective, target-specific siRNA design software for mammalian RNA interference, Yuki Naito et al., Nucleic Acids Res, 35 Vol 32, No Web Server issue Oxford University Press 2004). - "siRNA design tool" from Whitehead Institute of Biomedical Research at MIT http://jura.wi.mit.edu/pubint/http:/liona.wi.mit.edu/siRNAext/ - siRNA wizardTM from Invitrogen http: // www.sirnawizard.coml, Ambion's "siRNA Target Finder" http://www.ambion.com/techlib/misc/siRNA_finder.html, https://www.genscript.com/ssl-bin/app/rnai-http : //www.promega.com/siRNADesigner/default.htm -http: //bioweb.pasteur.fr/seganal/interfaces/sirna.html - Other programs are referenced on the site http://web.mit. edu / nuncmanus / www / homel.2files / siRNAs.htm, such as http: //athena.bioc.uvic.ca/cgi-binlemboss.pl?action=input&_app=sirna. The tools for siRNA preparation and cell transfection are available to the public by simple online ordering, such as the siRNA vectors commercialized by Invitrogen (http://www.invivogen.com/cat.php? ID = 3). Advantageously, the anti-MARK3 inhibitor is an interfering RNA that inhibits in vitro and / or in vivo the expression of a gene encoding a MARK3 protein. This RNAi is preferably chosen from antisense RNAs and double-stranded RNAs (dsRNAs), more preferably an siRNA. Interfering RNAs according to the invention are preferably designed to inhibit at least 50%, 75%, 90% or 95% or even more than 99% of the expression of a MARK3 protein in cells. According to a preferred embodiment of the invention, the siRNA comprises the following sequence, capable of inhibiting the expression of a gene encoding a MARK3 protein: Sequence sense: 5 'ACA GCA CUA UUC CUG AUC 3' The present invention also relates to a vector for the expression of an interfering RNA defined above and hereinafter, which vector comprises a sequence coding for said interfering RNA in the form of an anti-sense RNA. control of regulatory elements allowing expression of said interfering RNA in a host cell. Such vectors are known to those skilled in the art and are available, for example the pSilencerTM 5.1 Retro System 25 Ambions vectors (http://www.ambion.com/catalog/CatNum.php?5782) or Lentiviral RNAi BLOCK-iTTM. Expression Systems marketed by Invitrogen (https://catalog.invitrogen.com/index.cfm?fusioneaction = viewCatalog.viewProductDetails & productDexcription = 5449 & CMP = LEC-GCM S SEARCH & HQ S = block). The invention also relates to a vector for the delivery of an interfering RNA in a host cell, characterized in that it comprises an interfering RNA according to the invention, defined above and hereinafter, and means for delivering of said interfering RNA in a host cell. Such means are known to those skilled in the art, for example lipofectamines lipids (http://www.invitrogen.com/content.cfm?pageid=4005) or Mirus (http://www.mirusbio.com/). products / RNAi / index.asp).

  The invention also relates to an interfering RNA according to the invention, a vector for its expression or a vector for its delivery, as defined above and hereinafter, for their use in therapy. The present invention also relates to a pharmaceutical composition comprising an antibody according to the invention or an interfering RNA according to the invention or a vector for the expression of the interfering RNA according to the invention or a vector for the delivery of an interfering RNA according to the invention, as defined above and hereinafter, in a pharmaceutically acceptable vehicle. The methods for the administration of interfering RNA are known to those skilled in the art, the employed vehicles depending on the selected route of administration. Thus, IV injection of a chemically modified siRNA has been shown to be effective for inactivation of genes in vivo (Soutschek J, Akinc A, Bramlage B, Charisse K, Consist R, Donoghue M, Elbashir S, Geick A, Hadwiger P, Harborth J, John M, Kesavan V, Lavine G, Pandey RK, Racie T, Rajeev KG, Rohl I, Toudjarska I, Wang G, Wuschko S, Bumcrot D, Koteliansky V, Limmer S, Manoharan M, Vornlocher HP Of an endogenous gene by systemic administration of modified siRNAs.

  Nature. 2004, 432: 173-8). The invention also relates to the use of an antibody according to the invention or an interfering RNA according to the invention or a vector for the expression of the interfering RNA according to the invention or a vector for the delivery of an RNA interfering agent according to the invention, as defined above and hereinafter, for the treatment of cancers, more particularly glioblastomas, or for the preparation of a medicament for the treatment of said diseases. Those skilled in the art will be able to select the appropriate dosages according to the patient and the state of progress of the disease to be treated. This knowledge of the state of the patient and of the state of progress of the disease can advantageously be obtained by means of the diagnostic method according to the invention.

  It is understood that, according to the invention, the use can be done in combination with other therapeutic means suitable for the treatment of cancer, such as for example other drugs, cytotoxic molecules, antibodies or ligands that can be used. in oncology, but also means of treatment by radiation, in particular ionizing radiation or by surgery.

  In this case, the antibodies or interfering RNA according to the invention will be used in combination with the one or the other means simultaneously, together or separately, or shifted in time. In the case of staggered treatment, the antibodies or interfering RNA according to the invention may be used prior to the other therapeutic means or after the latter.

  The invention also relates to a variant of the MARK3 protein comprising the protein sequence of SEQ ID NO 1 and a nucleic acid sequence encoding said variant. It also relates to a vector for expressing a variant of the MARK3 protein comprising said nucleic acid sequence according to the invention under the control of regulatory elements necessary for the expression of said protein in a host organism. It also relates to a host organism comprising said expression vector according to the invention, and a method for preparing the variant of the MARK3 protein according to the invention, characterized in that it comprises the steps of culturing the host organism according to the invention in a suitable culture medium, then recovering the variant of the MARK3 protein thus produced and, optionally, its purification. Methods for cloning and expressing a protein in a host organism are well known to those skilled in the art, the regulatory elements constituting the vector being chosen by the latter according to the chosen host organism, but also according to the conditions of cultures and the objective of producing this variant of the MARK3 protein.

  One of these objectives can be the preparation and the implementation of a screening method of expression inhibitors of a MARK3 protein as defined previously or in the examples or of inhibitors of the activity of a MARK3 protein, the method comprising contacting at least one inhibitory candidate compound with an appropriate screening means to make it possible to demonstrate the activity of said compound with regard to the expression of MARK3 or its activity, the existence or the absence of inhibition. Such screening means are well known to those skilled in the art, such as, for example, a host organism expressing a reporter gene under the control of the MARK3 protein promoter or a host organism expressing the MARK3 protein, whose level of expression. or the transcription is controlled by appropriate methods, or a host organism expressing the MARK3 protein or a suitable reaction medium containing MARK3 protein, to control the activity of the latter under the effect of the candidate compound (s). According to one particular embodiment of the invention, several candidate compounds are tested, together or separately, said compounds being able to constitute a library of compounds to be tested. Advantageously, said compounds are chemical molecules called small molecules.

  The examples of embodiment below make it possible to better illustrate the invention without, however, seeking to limit its scope. List of examples 1. Demonstration of discriminant immunoreactive characteristics in biological fluids of populations of patients with tumors versus healthy individuals 2. Characterization by sequencing and mass spectrometry of the antigen responsible for discriminant immunoreactions 3. Identity of the protein antigenic 4. Western blot analysis of antigen-antibody reactions and validation of clinical interest 5. Impact of antibodies on viability of tumor cells in vitro 6. Blocking of tumor cell proliferation in vitro by siRNAs.

  DESCRIPTION OF THE FIGURES FIG. 1: Western blot made with a cystic fluid tested to demonstrate reactions against proteins of tumor origin. Three different samples were used to generate the protein fingerprints for the immuno-detection reaction after electrophoresis. Lane M: Protein Extract from Membranes of Glioblastoma Cells Lane C: Cytoplasmic Fraction of Glioblastoma Cells Track T: Total extract of glioblastoma cells. Figure 2: Sequence of the clone 2C10 cDNA (in A) and the expressed protein (in B).

  Figure 3: Alignment of the protein sequence expressed by clone 2C10 with the sequence of the C ter end of the protein isoform 4 of MARK3. Figure 4: Measurement of the intensity of the immunoreactions detected in the sera of different groups of individuals vis-à-vis the antigenic protein encoded by clone 2C10. Groups of individuals are composed of: group 1: normal individuals controls; group 2: patients with glioma responding positively to chemotherapy; group 3: glioma patients not responding to chemotherapy The immunoreactivity histograms correspond to the signals recorded in western blots (expressed in arbitrary units, AU) with the following antigens: A: total protein expressed by clone 2C 10 (corresponding to the variant domain of the MARK3 isoform 4 described in Example 3, FIG. 3); B: 11 kDa fragmentation product of the variant domain of the MARK3 isoform 4 described in Example 3. Figure 5: Measurement of cytotoxicity of purified immunoglobulins of cystic fluids or sera on tumor cells in culture. Figure 6: Sequences of siRNA tested in vitro.

  Example 1: Demonstration of Discriminatory Immunoreactive Characteristics in the Biological Fluids of Tumor Patient Populations Versus Healthy Individuals In this example, cystic fluids taken from CNS solid tumors of various patients were analyzed to determine if these liquids contain antibodies capable of recognizing proteins expressed by tumors. To do this, the reactivity of cystic fluids with respect to protein extracts of tumors was tested by Western blot. The western blot presented in FIG. 1 shows several colored bands, thereby demonstrating numerous reactivities, of modest or very intense intensity, with respect to various tumor proteins. For example, strong reactions to molecular weight proteins estimated at about 36, 41 and 53 kDa are noted. Given the protein fractions used in this western blot, it can be concluded that the tumor antigens against which the antibodies present in the cystic fluids are directed are located either in the membranes of the tumor cells or in their cytoplasm. After incubating western blot fingerprints with cystic fluid, antibodies that interacted with the antigenic proteins are detected by conventional methods using peroxidase-coupled secondary antibodies. The demonstration of these immunoreactions and antibodies directed against tumor proteins was continued according to the strategy described below. The analysis consisted in revealing the presence of antibodies, in the cystic fluids and also in the sera of the patients, responsible for the immune responses towards various human proteins. Immunoreaction tests of the sera and cystic fluids against a library of bacterial expression clones expressing various proteins of the human repertoire made it possible to isolate a clone named according to the nomenclature of the clone library proper to the inventors: clone 2C10. The biological fluids tested (cystic tumor fluids and sera from tumor patients) show a very strong reactivity towards the clone. Conversely, the sera of healthy individuals show a much lower reactivity or a virtual absence of reactivity. This experiment thus highlights the existence of antibodies directed against a particular human protein in the body fluids of healthy individuals or patients with tumors and the fact that the levels of these antibodies are different according to the groups of individuals tested. The measurement of these antibody levels therefore has the diagnostic value of making it easy to discriminate individuals with tumors from perfectly healthy individuals.

  Example 2 Characterization by Sequencing and Mass Spectrometry of the Antigen Responsible for Discriminatory Immunoreactions The protein which is expressed by clone 2C10 has been identified. This identification was carried out in two different and complementary ways. First, the characterization was based on sequence analysis of the cDNA cloned into the clone expression vector; finally, the human polypeptide expressed by this clone was purified and analyzed after trypsin proteolysis by means of a nanoLC-MS / MS approach (liquid nanochromatography coupled to tandem mass spectrometry analysis) (according to the protocol used by Bourges et al. al, 2004). The first approach was to extract the plasmid from the bacterial clone from the stock of the bank and cultured. Extraction was performed using standard bacterial plasmid purification methods (i.e., basically, by alkaline lysis and precipitation of plasmid DNA). The purified plasmids were then sequenced by the chain termination enzymatic technique with fluorescent dideoxynucleotides. The sequence was deciphered by capillary migration on an ABI 3700 sequencer. The sequences were made in the sense and antisense directions and validated by several runs. The use of software such as Autoassembler (Applied Biosystems) has made it possible to generate the consensus and integral sequence of the cDNA fragment cloned into the plasmid. The sequence has been studied in detail to recognize the regions of this sequence that encode the expressed human protein (so-called "coding" sequence). The cDNA sequence coding for clone 2C10 is shown in FIG. 2. FIG. 2 also shows the sequence of the human protein expressed by the clone as predicted from the cDNA sequence cloned into the expression vector. The sequence of the plasmid located upstream of the cDNA and comprising the codon of initiation of synthesis of the protein fragment encoded by the cDNA is not shown. The translation stop codon is shown in bold and underlined. The sequence of the protein expressed by the 2C10 expression plasmid is shown (in B). The conventional one-letter amino acid code is used. For clone 2C10, the specific amino acid sequence of the variant identified by the invention is underlined.

  Purification of the human protein expressed by the clone was performed. The experiments were conducted based on standard protocols for purification and manipulation of proteins. Thus, the bacterial clone 2C10 was cultured individually, then the bacterial cells were harvested and lysed in the presence of guanidinium salts. The human proteins were purified using affinity chromatography exploiting the interactions of the poly-histidine sequences with immobilized metal loaded resin columns. Indeed, because of the plasmid construction, the human proteins are expressed in the clones in the form of chimeras which integrate at their N-terminus a small sequence comprising six consecutive residues of histidines. This motif allows the quasi-selective retention of human proteins expressed in clones on Nickel chelating resins. After elution at acidic pH, the proteins were subjected to polyacrylamide gel electrophoresis. Then the major protein band is cut out and treated with trypsin. The peptides obtained are separated by reverse phase chromatography coupled with mass spectrometric analysis. This analysis makes it possible to obtain information on the primary sequence of the generated tryptic peptides. It thus makes it possible to unambiguously validate that the proteins encoded by the cDNAs cloned into the expression vectors are synthesized by the clones and constitute the antigens detected by immunoreaction.

  Clone 2C10 expresses the protein whose sequence is shown in FIG. 2.

  Example 3: Identity of the Antigenic Protein An in silico analysis consisting in comparing the cDNA sequence of clone 2C10 and the corresponding protein it expresses with respect to libraries of nucleic acid reference sequences (clone sequences). CDNA and human genome sequence) and human proteins was performed. This analysis made it possible to specify the identity of the protein which constitutes one of the specific antigens responsible for the immunoreactions shown in Example 1. The following information was thus obtained: Clone 2C expresses an unknown variant of a fragment of the MARK3 protein. The MARK3 protein is a protein kinase. The MARK3 gene is localized at 14q32.3. The sequence of the MARK3 fragment expressed by clone 2C10, which corresponds to the antigen responsible for the immunoreactivity of the biological fluids that is the subject of the invention, is identical to 84% with the known reference sequence of a MARK3 isoform (isoform 4 of MARK3 bearing the nomenclature P27448-4 in the Swiss-Prot database). It consists of the 279 C-terminal amino acids but includes an additional 52 amino acid sequence, located 215 amino acids upstream of the C-terminal amino acid of the reference sequence (see sequence underlined in Figure 2). Excluding this additional 52 amino acid sequence, the protein sequence expressed by clone 2C10 is 100% identical with the C ter end of MARK3 isoform 4. The protein sequence encoded by this clone is original and is not referenced in the Genbank library (http://www.ncbi.nlm.nih.gov/). The sequence of the cDNA fragment present in clone 2C10 was compared to the genomic sequence of the gene responsible for the expression of MARK3 in human tissues. This analysis revealed that the sequence of the original 52 amino acids present in the MARK3 variant described here is encoded by a 156-base cryptic exon that has been conserved during the splicing of the normal MARK3 transcript. The sequence of the protein expressed by the 2C10 clone is compared with the reference sequence of the MARK3 protein in Figure 3. The sequence identities are symbolized by stars.

  EXAMPLE 4 Western-blot Analysis of Antigen-antibody Reactions and Validation of Clinical Interest Immunodetection tests were undertaken to validate two important parameters: first, to check the specificity of the reactivity of the antibodies present; in sera against purified human antigenic proteins or fragments thereof; on the other hand, to evaluate the biological relevance of antibodies as indicators of diagnostic interest in oncology.

  These tests were performed using the Western-blot method. To do this, bacterial clone 2C10 was cultured, and the expressed human protein was purified on nickel chelator resin as shown in the previous example. The purified protein was electrophoretically migrated in polyacrylamide gel. After electrotransfer on PVDF membranes, the antigen is detected by impregnation of the membranes with various sera obtained from blood samples taken on many individuals. The cohort of individuals constituted for analysis was composed of 50 healthy individuals; 20 individuals with glial tumors who do not respond to chemotherapy (treatment with Temodal, or temozolomide from Schering Plow); and 14 individuals with glial tumors characterized by an objectified sensitivity to this chemotherapy (based on the regression of the tumor size observed at imaging at three-month intervals). The analysis of western blots led to the following conclusions. For clone 2C10, the sera react against two regions of the western blot, a region corresponding to an antigen of apparent size of 45 kDa and one corresponding to a size of 11 kDa. Enzymatic digestion analysis of the equivalent electrophoretic gel zones followed by nanoLC-MS / MS characterization shows that the 45 kDa zone corresponds to the original identified variant of the MARK3 protein expressed by the bacterial clone 2C10 and the zone of 11. kDa to a fragmentation peptide generated during the purification of the protein. This fragment encompasses the N-terminal portion of the antigen produced by clone 2C 10. The intensities of immunodetection reactions obtained on Western blots were quantified. Analysis of the data obtained individually with the sera of the 84 individuals in the cohort revealed that the response to the 45 kDa protein and the 11 kDa peptide was almost non-existent in the sera of the healthy individuals and that this response was intense with the sera of patients with glial tumors (without major distinction between tumors characterized by sensitivity to chemotherapy and those that are resistant). This example clearly shows that the detection of serum antibodies directed against the MARK3 protein has a diagnostic and prognostic value for the clinical management of patients with glial tumors. Indeed, the appearance of serum antibodies directed against the variant of the MARK3 protein (or its fragments) provides an obvious indication of the presence of solid tumors of the central nervous system. The results presented here show that particular epitopes present in the described protein (MARK3 variant) allow an evaluation of the changes in antibody levels in the body fluids of an individual; evaluation of major clinical interest. Any other biological or artificial, natural or chimeric component which comprises the epitopes recognized by the antibodies which are detected in the process described and are of clinical interest can be advantageously used to carry out assays in the spirit of the process described in this patent. . Figure 4 shows Western blot analysis of antigenic proteins produced by clone 2C10. Proteins and peptides resulting from spontaneous fragmentation were subjected to electrophoretic migration in polyacrylamide gel. The immunoreactions were conventionally revealed according to the western-blot approach after impregnation of blots with mixtures of sera from various groups of individuals. Groups of individuals are composed of: group 1: normal individuals controls; group 2: patients with glioma responding positively to chemotherapy; group 3: glioma patients not responding to chemotherapy The immunoreactivity histograms correspond to the signals recorded in western blots with the following antigens: A: total protein expressed by clone 2C10 (corresponding to the variant domain of the isoform 4 of MARK3 described in Example 3 (FIG. 3); B: 11 kDa fragmentation product of the variant domain of MARK3 isoform 4 described in Example 3, FIG.

  EXAMPLE 5 Impact of Antibodies on the Viability of Tumor Cells In Vitro In order to confirm the foreseeable therapeutic use of antibodies directed against the identified tumor antigens, the cytotoxic impact of immunoglobulins extracted from biological fluids on tumor cells in culture has been evaluated.

  The experiment consisted in preparing samples of purified immunoglobulins, extracted from the sera of healthy individuals and cystic fluids from individuals with a glial tumor. The immunoglobulins are purified by "Hitrap protein-G" column chromatography distributed by Amersham (General Electric). The conditions of use are strictly in accordance with those described by the supplier. The purified immunoglobulins are resuspended at a titre of 1 mg / ml. In vitro cultured tumor cells are contacted with the purified immunoglobulins at a final concentration of 1 microgram of immunoglobulins per ml in medium containing no fetal calf serum. The incubation is prolonged for 7 days. At the end of this incubation period, the viability of the cells is measured using a conventional cell viability test. The survival rates are calculated relative to controls corresponding to the cell cultures incubated with the preparation and dilution buffers of immunoglobulins but devoid of immunoglobulins (control buffer). Immunoglobulins were prepared from cystic fluids collected from tumors of the type: oligodendrogliomas or meningiomas or grade III astrocytoma. The cell types were represented by primary glioblastomas taken from different patients (2 different glioblastomas), an IMR32 neuroblastoma line, an EJ bladder carcinoma line. As shown in FIG. 5, the purified immunoglobulins of the sera of healthy individuals show very little cytotoxicity with respect to the different cell types. In contrast, purified immunoglobulins of cystic fluids of different origins significantly reduce the survival of glial tumor cells. The survival rate is between 15 and 40% depending on the extracts used. The cells of glioblastoma tumors are the most sensitive; bladder carcinoma cells are more modestly affected (65-75% survival); the viability of neuroblastoma cells is not significantly modified. This last element proves that at the concentration of 1 microgram of immunoglobulins per ml of medium, the purified immunoglobulins of cystic fluids do not show nonspecific toxicity to tumor cells in culture. The example makes it possible to conclude that immunoglobulins which are present in cystic fluids and which react with tumor antigens have an obvious cytotoxic capacity with respect to tumor cells. These antibodies are very clearly distinguishable from the immunoglobulins present in the sera of healthy individuals which do not show, as far as they are concerned, marked antitumor activity. The cytotoxic capacity of the purified immunoglobulins of cystic fluids is manifested not only to CNS tumor cells but also, albeit more modestly, to bladder cancer cells as indicated herein. The generalization of the use of these antibodies in therapeutic approaches therefore seems conceivable for the treatment of CNS tumors and certain forms of other cancers affecting organs other than the CNS. Figure 5 shows the cytotoxicity measurement of purified immunoglobulins of cystic fluids or sera on tumor cells in culture. The histograms represent the survival rates (expressed as arbitrary values) of the tumor cells in culture after 7 days of incubation in the presence of: 1) control buffer; 2) serum immunoglobulins from healthy individuals; 3) immunoglobulins of a cystic fluid of oligodendroglial tumor; 4) immunoglobulins of a cystic fluid of grade III astocytic tumor. The cells in culture are: in A) glioblastomas; in B) neuroblastomas. The concentrations of immunoglobulins in contact with the cells are established at 1 microgram of immunoglobulins per ml of medium in all the tests.

  Example 6 Blocking the In Vitro Proliferation of Tumor Cells with siRNAs The siRNA sequence making it possible to disrupt the expression of MARK3 proteins was chosen as a function of the cDNA sequence of clone 2C10 (see FIG. 2). Briefly, two sequences are selected for the creation of siRNA, namely: the sense sequence which has a length of 19 bases homologous to a portion of the messenger RNA sequence encoding the protein; and a perfectly complementary sequence of the selected "sense" sequence. Both sequences are 19 bases long. The sense and antisense sequences are shown in FIG. 6. The RNA fragments corresponding to sense sequences and complementary sequences were synthesized chemically. Double-stranded RNAs were then created in vitro by hybridization between the RNAs corresponding to the sense sequences and the fragments corresponding to the complementary sequences. These double-stranded RNA fragments were used for transfection of tumor cells in culture. The transfecting agent is here oligofectamine. Appropriate controls were also performed, in particular: transfection of cells according to a strictly identical protocol but not including siRNAs. The cells used were glioblastoma cells (U373 line showing a strong proliferation in vitro).

  U373 glioblastoma cells in culture were transfected with the siRNAs whose sequences are shown in FIG. 6. After 5 days of maintenance in a growth medium, the cells are counted and the proliferation rate, calculated with respect to initial seeding of the medium, is measured. Compared with normal cell development under control conditions (100% proliferation rate), the double-stranded siRNA directed against MARK3 significantly slows the proliferation of glioblastoma cells. Indeed, in the presence of the siRNA directed against MARK3, the proliferation of the cells is only 74% compared to the control.

  References - Beghini A, Magnani I, Roversi G, Piepoli T, Di Terlizzi S, Moroni RF, Polio B, Fuhrman Conti AM, Cowell JK, Finocchiaro G, Larizza L. Oncogene. 2003, 22, 2581-91. Blomberg K, Hurskainen P., Hemmila I. Clin Chem. 1999, 45, 855-61.

  JC Booth, Hannington G, Bakir TM, H Stem, Kangro H, Griffiths PD, Heath RB. J Clin Pathol. 1982, 35, 1345-8. Bourges I, Ramus C, Monsoon of Camaret B, Beugnot R, Remacle C, Cardol P, Hofhaus G, Issartel W. Biochem. J. 2004, 383, 491-9. - Bruner JM, Inouye L, Fuller GN, Langford LA. Cancer. 1997, 79, 796-803. Burnette WN., Anal Biochem. 1981, 112, 195-203. Cairncross JG, Ueki K, Zlatescu MC, Lisle DK, Finkelstein DM, Hammond RR, Silver JS, Stark PC, Macdonald DR, Ino Y, Ramsay DA, Louis DN. J Natl Cancer Inst. 1998, 90, 1473-9. Chatel M, Brucher J-M. in Neuro-Oncology, Publisher J. Hildebrand, Doin 2001; p 25-34 - SW Coons, Johnson PC, Scheithauer BW, AJ Yates, Pearl DK. Cancer. 1997, 79, 1381-93. - Ditzel HJ, Masaki Y, Nielsen H, Farnaes L, Burton DR. Proc Natl Acad Sci U S 2000, 97, 9234-9. - Daumas-Duport C, Figarella-Branger D., in Histopronostic Classification of Glial Brain Tumors and Their Impacts in Therapeutics Editions Spaces 34, 2002; p 11-48. Ejiri SI. Biosci. Biotechnol. Biochem. 2002, 66, 1-21. - Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Nature. 2001, 411, 494-8. Ellington AD, Szostak JW. Nature 1990, 346, 818-22. Engvall E, Perlmann P. Immunochemistry 1971, 8, 871-874. Engvall E, Jonsson K., Perlmann P. Biochim Biophys Acta. 1971, 251, 427-34. - Engvall E, Perlmann P.J Immunol. 1972, 109, 129-35. - Enomoto K, Aono Y, Mitsugi T, Takahashi K, Suzuki R, Preaudat M, Mathis G, Kominami G, Takemoto H. J Biomol Screen. 2000, 5, 263-8. - Fagerstam LG, Frostell A, Karlsson R, Kullman M, Larsson A, Malmqvist M, Butt H. J Mol Recognit. 1990, 3, 208-14. - Gao C, Mao S, CH Lo, Wirsching P, Lerner RA, Janda KD. Proc Natl Acad Sci U S 1999, 96, 6025-30. - Grant AG, RM Flomen, Tizard ML, Grant DA. Int J Cancer. 1992, 50, 740-5. Hechemy K, Stevens RW, Gaafar HA., Appl Microbiol. 1974, 28, 306-11.

  Hulett HR, WA Bonner, Barrett J, Herzenberg LA. Science. 1969, 166, 747-9. Karagiannis TC, El-Osta A. Cancer Gene Ther. 2005 May 13 Khalyfa A, Carlson BM, JA Carlson, Wang E. Dev Dyn. 1999, 216, 267-73. Kato T, Satoh S, Okabe H, Kitahara O, Ono K, Kihara C, Tanaka T, Tsunoda T, Yamaoka Y, Nakamura Y, Furukawa Y. Neoplasia. 2001, 3, 4-9. - Kiernan JA. Histological and histochemical methods. Theory and practice. Third edition. Oxford: Butterworth-Heinemann 1999. Knappik A, Ge L, Honegger A, Pack P, Fischer M, Wellnhofer G, Hoess A, Wolle J, Pluckthun A, Virnekas B. J Mol Biol 2000, 296, 57-86. - Kusnezow W, Hoheisel J, .D. J Mol Recognit. 2003, 16, 165-76. Laemmli UK. Nature. 1970, 227, 680-5. Lee JM. Reprod. Biol. Endocrinol. 2003, 1, 69. Mansilla F, Hansen LL, Jakobsen H, Kjeldgaard NO, Clark I3F, Knudsen CR. Biochim Biophys Acta. 2005, 1727, 116-24. - Mathis G. Clin Chem. 1995, 41, 1391-7. Merchant M, Weinberger SR. Electrophoresis. 2000, 21, 1164-77. - Mittler MA, Walters BC, Stopa EG. J Neurosurg. 1996, 85, 1091-4. - Ohkouchi K, Mizutani H, Tanaka M, Takahashi M, Nakashima K, Shimizu M. Int Immunol 1999, 11, 1635-40. Parks DR, Herzenberg LA. Methods Enzymol. 1984, 108, 197-241. Peluso P, Wilson DS, DoD, Tran H, Venkatasubbaiah M, Quincy D, Heidecker B, Poindexter K, Tolani N, Phelan M, Witte K, Jung LS, Wagner P, Nock S. Anal Biochem. 2003, 312, 113-24. - Shen R, ZZ Su., Olsson CA, Fisher PB. Proc Natl Acad Sci U S A 1995, 92, 6778-82. 25 - Singer JM, CM Plotz, Pader E, Elster SK. Am J Clin Pathol. 1957, 28, 611-7. - Szabo A, Stolz L., Granzow R. Curr Opin Struct Biol. 1995, 5, 699-705. Szollosi J, Damjanovich S, Matyus L. Cytometry. 1998, 34, 159-79. Towbin H, Staehelin T, Gordon J. Proc Natl Acad Sci U S 1979, 76, 4350-4. - Tuerk C, Gold L. Science 1990, 249, 505-10. Ueda H, Kubota K, Wang Y, Tsumoto K, Mahoney W, Kumagai I, Nagamune T. Biotechniques. 1999, 27, 738-42. Vikinge TP, Askendal A, Liedberg B, Lindahl T, Tengvall Biosens Bioelectron. 1998, 13,

  1257-62. Weinberger SR, Morris TS, Pawlak M. Pharmacogenomics 2000, 1, 395-416. Xu Y, DW Piston, Johnson CH. Proc Natl Acad Sci U S 1999, 96, 151-6. - Yalow RS, Berson SA. J Clin Invest. 1960, 39, 1157-75.

Claims (19)

  1. A method that can be used to detect the presence or absence of a tumor in a mammal, characterized in that it comprises a step of detecting and / or quantifying on a biological sample previously taken from said mammal: the presence of a MARK3 protein, and / or the presence of antibodies directed against a MARK3 protein or a fragment comprising at least one epitope of this protein.   2. Method according to claim 1, characterized in that the biological sample 10 previously taken is selected from a sample of blood serum, lymph, cystic fluid, cerebrospinal fluid (CSF), tissue homogenates, preferably a sample of blood serum,   3. Method according to one of claims 1 or 2, characterized in that it further comprises the detection and quantification of at least one other biological marker characteristic of the presence and / or invasiveness of a tumor.   4. Method according to one of claims 1 to 3, characterized in that the presence of the MARK3 protein is detected and / or quantified by means of antibodies directed against the MARK3 protein or at least one epitope of this protein.   5. Method according to claim 4, characterized in that the antibodies are polyclonal antibodies or monoclonal antibodies.   6. Method according to one of claims 1 to 3, characterized in that the presence of antibodies directed against a MARK3 protein or at least one epitope of this protein is detected and / or quantified using an antigen comprising at least an epitope of a MARK3 protein. 25   7. Method according to one of claims 1 to 6, characterized in that it comprises a step of comparing the results obtained at the step of detection and / or quantification with a reference value characteristic of the presence of a tumor and / or with a reference value characteristic of the absence of tumor.   8. Method according to one of claims 1 to 7, characterized in that the MARK3 protein is a variant comprising the protein sequence shown in SEQ ID NO 1.   9. Diagnostic kit for carrying out a method according to one of claims 1 to 8, characterized in that it comprises means for detecting and / or quantifying on a biological sample previously taken: - the presence of a MARK3 protein, and / or the presence of antibodies directed against a MARK3 protein or a fragment comprising at least one epitope of this protein.   10. Antibodies directed against a MARK3 protein or against a fragment comprising at least one epitope of this protein, characterized in that they bind specifically to a MARK3 protein or to at least one epitope of this protein.   11. A method of inhibiting the growth of tumor cells in vitro, characterized in that the activity of the MARK3 protein is inhibited by an antibody or interfering RNA that inhibits the expression of a gene encoding a MARK3 protein.   12. Interfering RNA characterized in that it inhibits in vitro and / or in vivo the expression of a gene encoding a MARK3 protein.   13. Interfering RNA according to claim 12, characterized in that it is selected from antisense RNA and double-stranded RNA (dsRNA).   Interfering RNA according to claim 13, characterized in that the double-stranded RNA is a siRNA.   Interfering RNA according to claim 14, characterized in that it comprises the following sequence, capable of inhibiting the expression of a gene encoding a specific variant of the MARK3 protein: Sequence sense: 5 'ACA GCA CUA UUC CUG AUC A 3 'Antisense sequence: 5' UGA UCA GGA AUA GUG U CUG 3 '   16. Vector for the expression of an interfering RNA according to one of claims 12 to 15, characterized in that it comprises a sequence coding for said interfering RNA under the control of regulatory elements allowing the expression of said RNA. interfering in a host cell. 20   17. Vector for the delivery of an interfering RNA in a host cell, characterized in that it comprises an interfering RNA according to one of claims 12 to 15 and means for the delivery of said interfering RNA in a host cell.   18. Pharmaceutical composition characterized in that it comprises an antibody according to claim 10 or an interfering RNA according to one of claims 12 to 15 or a vector for the expression of the interfering RNA according to claim 16 or a vector for delivery of an interfering RNA according to claim 17 in a pharmaceutically acceptable carrier. One of claims 12 to 15 for the preparation of a medicament for the treatment of cancers. 22. Use according to claim 21 for the treatment of glioblastomas. 23. Variant of the MARK3 protein comprising the protein sequence of SEQ ID NO 1.   19. interfering RNA according to one of claims 12 to 15 for its use in therapy. Antibody according to claim 10 for use in therapy.20. 21. Use of an antibody according to claim 10 or an interfering RNA according to 24. A nucleic acid sequence encoding a variant of the MARK3 protein according to claim 23. 25. An expression vector of a variant of the MARK3 protein comprising a nucleic acid sequence according to claim 24 under the control of elements of 5 necessary for the expression of said protein in a host organism. 26. Host organ comprising an expression vector according to claim 25. 27. Process for the preparation of a variant of the MARK3 protein according to claim 23, characterized in that it comprises the stages of cultivation of a host organism according to claim 26 in a suitable culture medium, then recovering the variant of the MARK3 protein so produced and, optionally, its purification.