Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

• the present invention relates to a new method for demonstrating the presence or absence of markers associated with tumors and their sensitivity to chemotherapy.
• the invention also relates to diagnostic kits comprising the means for implementing 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.
• diagnostic kits comprising the means for implementing 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.
• CNS 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.
• the present invention therefore relates to a new diagnostic method for detecting the presence or absence of a tumor and its sensitivity to chemotherapies in a mammal, in particular in humans, by detecting and / or quantifying the presence of a tumor.
• new biological marker in a biological sample previously taken from said mammal an eEF1Al protein (elongation factor of protein synthesis, Swiss-Prot reference: http://www.expasy.org/uniprot/P68104).
• eEF1A1 protein or antibodies directed against an eEF1A1 protein or a fragment comprising at least one epitope of said protein, or a low concentration relative to the concentrations observed in healthy persons or patients with chemotherapies is characteristic of the presence of a tumor that is a priori resistant to the usual chemotherapies (chemoresistant).
• a level of eEF1A1 protein or of antibodies directed against an eEF1A1 protein or a fragment comprising at least one epitope of said protein comparable to that observed for healthy persons is characteristic of a tumor that is a priori sensitive to the usual chemotherapies. (chemo).
• the present invention also relates to a new 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.
• PAR-I is a Disheveled-associated kinase and a positive regulator of signaling.
• 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 therefore relates to a method that can be used to detect the presence or absence of a tumor and / or its sensitivity to chemotherapies 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 an eEF1A1 protein, and / or the presence of antibodies directed against an eEF1A1 protein or a fragment comprising at least one epitope of this protein, and / or 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.
• eEF1A1 protein is intended to mean a protein which comprises a protein sequence of the reference eEF1Al protein (Swiss-Prot: http://www.expasy.org/uniprot/P68104), its isoforms, its variants and its biologically active fragments.
• MARK3 protein is intended to mean a protein which comprises a protein sequence of the reference MARK3 protein, described in reference GenBank accession number.
• the isoforms, fragments or biologically active variants according to the invention are recognized by the anti-eEF1A1 or anti-MARK3 antibodies respectively.
• variants is intended to mean proteins which advantageously have at least 75% identity with the reference eEF1Al protein or with the reference MARK3 protein, respectively, 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.gov/BLAST/ 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).
• CLUSTALW programs http: // www. with the default settings on these sites.
• Differences between the eEF1A1 or MARK3 variants and their respective 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 reference sequence. The differences may also be due to mutations, with at least one amino acid being substituted for a different amino acid in the reference sequence. The differences may also be due to the addition of at least one amino acid within the reference sequence. When multiple acids Amines are added within the reference sequence, they may be contiguous, i.e. forming a protein fragment of at least 2 amino acids, or distributed over the sequence or both.
• the eEF1A1 variant comprises at least one protein fragment inserted into the reference eEF1Al sequence. This fragment can comprise up to 100 amino acids, generally between 10 and 60 amino acids.
• the eEF1A1 protein comprises the protein sequence represented in SEQ ID NO 1.
• the MARK3 variant comprises at least one protein fragment inserted into the reference MARK3 sequence.
• This fragment can comprise up to 100 amino acids, generally between 10 and 60 amino acids.
• the MARK3 protein is a variant comprising the protein sequence represented in SEQ ID NO 5.
• the biological sample previously taken is selected from a sample of blood serum, lymph, cystic fluid, tissue homogenates, preferably a sample of blood serum.
• the biological sample taken may be subjected to a prior treatment for its analysis, for example grinding and / or dissolution.
• 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.
• an extract containing at least one antigen according to the invention is subjected to electrophoretic migration in polyacrylamide gel in denaturing medium.
• This technique is well known in one of these variants under the acronym of SPAGE: for "SDS 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.
• 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.
• 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.
• ELISA tests can be performed on devices such as the VIDAS system distributed by Bio-Mérieux c) methods based on "protein microarrays"
• 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.) containing antigens. fixed by various chemical methods on the support, each antigen being deposited on a small surface of the support representing a few micron-square area. 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).
• the immune complexes formed 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.
• 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 BIAcore (BIAcore AB, Uppsala, Sweden, http://www.biacore.com).
• the detection of the antibodies can be carried out using, for example, so-called SELDI -TOF technology (Merchant and Weinberger, 2000, Weinberger et al, 2000). This embodiment is carried out by exploiting the technical platform distributed by Ciphergen (Ciphergen Biosystems, Inc. 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.
• 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.
• the antigens of interest are chemically coupled to particulate components of micrometric sizes such as colored or non-colored 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 is reflected by the formation of bundles of logs whose size becomes macroscopically important to the point of being "visible" to the eye by an operator.
• 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).
• 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 RIA principle are distributed, for example, for various assays 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.
• FACS Fluorescent Activated Cell Sorting
• a fluorescently labeled reagent may be used to label whole cells derived from and isolated from biopsies and to allow sorting and quantification positive cells for the presence of the desired antigens.
• 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 detects individually the intensity of the fluorescent signal associated with each cell, and counts the number of cells detected and optionally isolates the cells in cells. specific tanks.
• 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 called Becton Dickinson's "Bead Array Cytometry" (http://www.bdeurope.com); or the Luminex system from Miraibio (www: //www.miraibio.com).
• 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.
• 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). Protein analysis by the fluorescence transfer method can be performed on the Kryptor® apparatus of the German company BRAHMS (www.brahms.de).
• the fluorescent compound excited by the excitation light radiation in the FRET technique can be substituted by a bioluminescent system which relies on the activity of an enzyme (Xu et al, 1999)
• a bioluminescent system which relies on the activity of an enzyme
• 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 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.
• the assay of the antigens themselves or their fragments or metabolic modification products may also constitute an analytical application of clinical interest.
• reagents 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 that may interact 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 GoId 1990).
• antigens phages or recombinant bacteria expressing on their surface polypeptide regions capable of interacting with haptens or antigens
• aptamers chemical molecules of the polynucleotide or even polypeptide type capable of establishing high affinity non-covalent interactions with target molecules
• 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 in catalogs, for example in the Sigma Aldrich catalog, available online (http://www.sigmaaldrich.com).
• the presence of the eEF1A1 protein or the MARK3 protein is detected and / or quantified by means of antibodies directed against the eEF1A1 protein or against the MARK3 protein, respectively, or at least one epitope of one of these proteins.
• the antibodies are advantageously chosen from polyclonal antibodies or monoclonal antibodies.
• the antigen comprises an eEF1Al protein as defined above and hereinafter.
• the antigen may comprise simple fragments of an eEF1A1 protein, it being understood that said fragment comprises at least one epitope recognized by the anti-eEF1A1 antibodies.
• the antigen comprises a MARK3 protein as defined above and hereinafter.
• 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.
• 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.
• 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.).
• the level of recognition between the epitope and the different antibodies may vary from one epitope-antibody pair to another.
• 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.
• 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.
• 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.
• a fragment comprising at least one epitope of the eEF1A1 protein is, for example, a fragment encompassing the N-terminal part of the eEF1A1 protein, more particularly a 12-kD fragment encompassing this N-terminal part.
• 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-kD fragment encompassing this N-terminal portion.
• 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 chemoresistant tumor and / or with a value of reference characteristic of the presence of a chemosensitive tumor.
• eEF1A1 or anti-eEF1A1 antibodies may be different depending on the means used for the detection and / or quantification of eEF1A1 or anti-eEF1A1 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 individuals known to be carriers of tumors on the other hand, with in this second population the distinction between individuals known to have a chemosensitive tumor and those known to have a chemoresistant tumor.
• the anti-MARK3 inhibitor specifically inhibits the MARK3 variant, the protein sequence of which is represented in SEQ ID No. 5. Specific inhibition is understood according to the invention. it inhibits the target variant, without substantially inhibiting the other variants of MARK3.
• 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.
• at least one other biological marker characteristic of the presence and / or invasiveness of a tumor and / or its chemoresensitivity may be detected and quantification of at least one other biological marker characteristic of the presence and / or invasiveness of a tumor and / or its chemoresensitivity.
• the KI67 antigen protein as proliferation marker (SwissProt reference: hrtp: / ⁇ www.expasy.org/'uniprut/P46 ⁇ i3), or else the phosphorylated vimentin, the absence of which is characteristic of an invasive tumor (PCT / EP2005 / 054598 filed on September 15, 2005)
• the method comprises a step of detecting and / or quantifying on a biological sample previously taken at a time: the presence of an eEF1A1 protein, and / or the presence of antibodies directed against an eEF1A1 protein or a fragment comprising at least one epitope of this protein, and 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.
• 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 taken: the presence of an eEF1A1 protein, and / or the presence of antibodies directed against an eEF1A1 protein or a fragment comprising at least one epitope of this protein and / or the presence of a MARK3 protein, and / or the presence of antibodies directed against an MARK3 protein or a fragment comprising at least one epitope of this protein.
• eEF1Al or MARK3 antigen respectively defined above, or an anti-eEF1Al or anti-MARK3 antibody according to the invention and reagents necessary for the implementation of the diagnostic method according to the invention.
• the detection kit comprises a suitable support, able to receive the biological sample and the appropriate detection means.
• 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.
• suitable means for example a covalent bond or adsorbed on the support.
• the present invention also relates to antibodies directed against an eEF1Al protein or against a MARK3 protein or against a fragment comprising at least one epitope of one of these proteins, which specifically bind to an eEF1Al protein or a MARK3 protein, respectively , or at least one epitope of one of these proteins defined above and hereinafter.
• the invention also relates to said antibodies for their use in therapy.
• eEF1Al or MARK3 as markers of the presence of tumor cells in mammals, in particular in humans, and where appropriate their sensitivity to chemotherapies
• the inventors have also found that the inhibition of activity or expression of eEF1A1 or 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 the activity of the eEF1A1 protein or the MARK3 protein is inhibited by means of an anti-eEF1Al inhibitor or anti-MARK3 respectively.
• the anti-MARK3 inhibitor specifically inhibits the variant of MARK3, the protein sequence of which is represented in SEQ ID No. 5. Specific inhibition is understood according to the invention. it inhibits the target variant, without substantially inhibiting the other variants of MARK3.
• the anti-eEF1Al or anti-MARK3 inhibitors inhibiting the activity of eEF1A1 or MARK3 respectively act on the eEF1A1 protein or the MARK3 protein, respectively, preventing or limiting its capacity to exert its biological function. It may be for example an antibody whose antigen is an eEF1A1 protein or a MARK3 protein respectively, or at least one epitope of one of these proteins, as defined above and hereinafter.
• the anti-eEF1A1 inhibitors inhibiting the expression of eEF1A1 or the MARK3 inhibitors inhibiting the expression of MARK3, act at the level of the transcription of the gene coding for eEF1A1 or MARK3 respectively or at the level of the translation of the RNA towards the protein.
• it may be an interfering RNA which hybridises to the messenger RNA (mRNA) gene expression product comprising the sequence coding for the eEF1A1 protein or, respectively, for the MARK3 protein to inhibit translation, either by simple steric hindrance or to promote the cleavage of the mRNA.
• mRNA messenger RNA
• the interfering RNAs are prepared and selected to obtain at least
• 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.
• siRNAs design and preparation of siRNAs and their use for in vivo and in vitro cell transfection are well known and widely described in many publications, such as that: 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
• 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
• siRNA wizard TM from Invitrogen http: // www. simawizard.com/,
• siRNA preparation and cell transfection are available to the public by simple on-line ordering, such as the siRNA vectors marketed by Invitrogen (bttpj // wwwjn). ⁇
• the anti-eEF1A1 inhibitor or the anti-MARK3 inhibitor is an interfering RNA that inhibits, in vitro and / or in vivo, the expression of a gene encoding an eEF1A1 protein or encoding a MARK3 protein, respectively.
• This RNAi is preferably chosen from antisense RNAs and double-stranded RNAs (dsRNAs), more preferably an siRNA.
• the 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 an eEF1A1 protein or a MARK3 protein in the cells. .
• the siRNA comprises the following sequence, capable of inhibiting the expression of a gene encoding an eEF1 Al protein: Sequence sense: 5 'UGG UGA CAA CAU GCU GGA G 3' Antisense sequence): 5 'CUC CAG CAU GUU GUC ACC A 3'
• 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 A 3'
• Antisense sequence 5 'UGA UGA GGA AUA GUG CUG U 3'
• the siRNA comprises the following sequence, capable of inhibiting the expression of a gene coding for the specific variant of the MARK3 protein (SEQ ID NO 5)
• Antisense sequence 5 'CUU-CAC-UGU-CUA-UUG-GAG-G 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 under the control of regulatory elements allowing the expression of said interfering RNA. in a host cell.
• vectors are known to those skilled in the art and are available, for example the Ambions vectors pSilencer TM 5.1 Retro System
• 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 the delivery of said RNA interfering in a host cell.
• 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
• 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 interfering RNA according to the invention or a vector for the delivery of an interfering RNA according to the invention. as defined above and hereafter in a pharmaceutically acceptable carrier.
• 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.
• IV injection of a chemically modified siRNA has been shown to be effective for inactivation of genes in vivo (Soutschek
• 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.
• 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.
• 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.
• the antibodies or interfering RNA according to the invention will be used in combination with the one or more other means simultaneously, together or separately, or shifted in time.
• 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 eEF1A1 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 eEF1A1 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 eEF1A1 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 eEF1A1 protein thus produced and, optionally, its purification.
• the invention also relates to a variant of the MARK3 protein comprising the protein sequence of SEQ ID NO 5 and a nucleic acid sequence encoding said variant.
• She also relates to an expression vector of 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 the production of this variant of the eEF1Al protein or MARK3 protein respectively.
• One of these objectives can be the preparation and the implementation of a method for screening expression inhibitors of an eEF1Al protein or a MARK3 protein, as defined previously or in the examples, or inhibitors of the activity of an eEF1A1 protein or 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 at look at the expression of eEFlAl or respectively of MARK3, or their respective activity, the existence or the absence of an 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 promoter of the eEF1A1 protein or, respectively, MARK3 or a host organism expressing the eEF1A1 protein, or, respectively, MARK3. , whose level of expression or transcription is controlled by appropriate methods, or a host organism expressing the eEF1A1 protein or MARK3 respectively or a suitable reaction medium containing eEF1A1 protein, or MARK3 respectively, to control the activity of said protein under the effect of the candidate compound (s).
• candidate compounds are tested, together or separately, said compounds being able to constitute a library of compounds to be tested.
• said compounds are chemical molecules called "small molecules”.
• the selected compounds specifically inhibit the expression or the variant activity of MARK3, the protein sequence of which is represented in SEQ ID No. 5.
• 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 C Cytoplasmic fraction of glioblastoma cells Track T: Total extract of glioblastoma cells.
• Figure 2 Sequence of the cDNA clone 1G5 (in A) and the expressed protein (in B).
• Figure 3 Alignment of the protein sequence expressed by the clone 1G5 with the sequence of the C ter end of the protein isoform 4 of eEFlA1.
• 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 the clone 1G5.
• Groups of individuals are composed of: group 1: normal individuals controls; group 2: patients with glioma responding positively to chemotherapy; group 3: patients with glioma not responding to chemotherapy
• Histograms of immunoreactivity correspond to signals recorded in western blots (expressed in arbitrary units, AU) with the following antigens:
• A total protein expressed by clone 1G5 (corresponding to the domain of the variant of isoform 4 of eEF1A1 described in Example 3, FIG. 3);
• Figure 5 Cytotoxicity measurement of purified immunoglobulins of cystic fluids or sera on tumor cells in culture.
• Figure 6 Sequences of siRNA tested in vitro.
• Figure 8 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 of glioblastoma cell membranes
• Lane C Cytoplasmic fraction of glioblastoma cells
• Track T total extract of glioblastoma cells.
• Figure 9 Sequence of the clone 2C10 cDNA (in A) and the expressed protein (in B).
• Figure 10 Alignment of the protein sequence expressed by the clone 2C10 with the sequence of the C ter end of the protein isoform 4 of MARK3.
• Figure 11 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: patients with glioma not responding to chemotherapy
• Histograms of immunoreactivity correspond to signals recorded in western blots (expressed in arbitrary units, AU) with the following antigens:
• A total protein expressed by the 2C10 clone (corresponding to the variant domain of the MARK3 isoform 4 described in Example 9, FIG. 10); B: HkDa fragmentation product of the variant domain of the MARK3 isoform 4 described in Example 9.
• Figure 12 Measurement of cytotoxicity of purified immunoglobulins of cystic fluids or sera on tumor cells in culture.
• Figure 13 Sequences of siRNAs directed against MARK3 and its isoform 4 tested in vitro.
• Figure 14 Measurement of the cytotoxicity of siRNAs directed against MARK3 and its isoform 4 on tumor cells in culture.
• EXAMPLE 1 Demonstration of Discriminatory Immunoreactive Characteristics in the Biological Fluids of Populations of Patients with Tumors versus Healthy Individuals
• cystic fluids collected from CNS solid tumors of various patients were analyzed for antibodies containing antibodies that can recognize tumor-expressed proteins.
• 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 highlighting 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.
• Example 2 Characterization by Sequencing and Mass Spectrometry of the Antigen responsible for Discriminatory Immunoreactions
• the protein which is expressed by the 1G5 clone has been identified. This identification was carried out in two different and complementary ways.
• the characterization was based on sequence analysis of the cloned cDNA in the clone expression vector; finally, the human polypeptide expressed by this clone has been purified and analyzed after proteolysis with trypsin using a nanoLC-MS / MS approach (liquid nano-chromatography coupled with tandem mass spectrometry analysis) (according to the protocol used by Bourges et 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) made it possible to generate the consensus and integral sequence of the cloned cDNA fragment in the plasmid.
• Autoassembler Applied Biosystems
• FIG. 2 also shows the sequence of the human protein expressed by the clone as predicted from the sequence of the cDNA cloned in the vector of expression.
• 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 expression plasmid 1G5 is shown (in
• 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 1G5 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.
• sequence of the protein expressed by the 1G5 clone is compared with the reference sequence of the eEF1A1 protein in Fig. 3.
• sequence identities are symbolized by stars.
• Example 4 Western blot analysis of antigen-antibody reactions and validation of clinical interest
• Immunodetection tests have been undertaken to validate two important parameters: first, to check the specificity of the reactivity of the antibodies present in the sera with respect to the purified antigenic human proteins or their fragments; on the other hand, to evaluate the biological relevance of antibodies as indicators of diagnostic interest in oncology.
• the cohort of individuals constituted for the 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).
• Figure 4 shows Western blot analysis of antigenic proteins produced by clone 1G5. 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: patients with glioma not responding to chemotherapy
• Histograms of immunoreactivity correspond to the signals recorded in western blots with the following antigens:
• A total protein expressed by clone 1G5 (corresponding to the sequence of eEF1A described in example 3, FIG. 3); B: 12kDa fragmentation product of eEF1A1 described in Example 3, FIG. EXAMPLE 5 Impact of the Antibodies on the Viability of the Tumor Cells In Vitro
• 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 immunoglobulin 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.
• the purified immunoglobulins of the sera of healthy individuals show very little cytotoxicity with respect to the different cell types.
• 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 any nonspecific toxicity with respect to tumor cells in culture.
• 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 purified immunoglobulins of cystic fluids is manifested not only to CNS tumor cells but also, more modestly, to bladder cancer cells, as indicated right here.
• 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
• 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.
• siRNA sequence making it possible to disrupt the expression of eEF1A1 proteins was chosen as a function of the cDNA sequence of clone 1G5 (see FIG. 2).
• siRNA 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.
• 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 to normal cell development under the control conditions (100% proliferation rate), the double-stranded siRNA directed against eEF1A1 significantly slows the proliferation of glioblastoma cells.
• the “Middle” condition corresponds to the control without transfection.
• the “GFP” conditions correspond to a transfection with a siRNA directed against the GFP protein (transfection safety controls).
• Example 7 Demonstration of Discriminatory Immunoreactive Characteristics in the Biological Fluids of Tumor Patient Populations Versus Healthy Individuals
• cystic fluids collected from CNS solid tumors of various patients were analyzed to determine whether these fluids contain antibodies that can recognize proteins expressed by tumors.
• the reactivity of cystic fluids with respect to protein extracts of tumors was tested by Western blot.
• the western blot shown in FIG. 8 shows several colored bands, thereby highlighting 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.
• the protein that 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 cloned cDNA in the clone expression vector; finally, the human polypeptide expressed by this clone was purified and analyzed after trypsin proteolysis using a nanoLC-MS / MS approach (liquid nano-chromatography coupled to tandem mass spectrometry analysis) (according to the protocol used by Bourges) et 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) made it possible to generate the consensus and integral sequence of the cloned cDNA fragment in 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").
• FIG. 9 also shows the sequence of the human protein expressed by the clone as predicted from the sequence of the cDNA cloned into the cloning vector. expression.
• 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.
• the specific amino acid sequence of the variant identified by the invention is underlined.
• 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 pattern allows the quasi-selective retention of proteins expressed in clones on Nickel chelating resins.
• 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 in 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. 9.
• Clone 2C10 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).
• Immunodetection tests have been undertaken to validate two important parameters: first, to check the specificity of the reactivity of the antibodies present in the sera with respect to the purified antigenic human proteins or their fragments; on the other hand, to evaluate the biological relevance of antibodies as indicators of diagnostic interest in oncology.
• the cohort of individuals constituted for the 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).
• Figure 11 shows the 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: patients with glioma not responding to chemotherapy
• Histograms of immunoreactivity correspond to the signals recorded in western blots with the following antigens:
• A total protein expressed by the 2C10 clone (corresponding to the variant domain of the MARK3 isoform 4 described in Example 9, FIG. 10); B: HkDa fragmentation product of the variant domain of the MARK3 isoform 4 described in Example 9 Figure 10.
• the cytotoxic impact of the immunoglobulins extracted from the biological fluids on the tumor cells in culture was 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 immunoglobulin per ml in medium containing no fetal calf serum. The incubation is prolonged for 7 days.
• 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.
• the purified immunoglobulins of the sera of healthy individuals show very little cytotoxicity to different cell types.
• 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 any nonspecific toxicity with respect to tumor cells in culture.
• Figure 12 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.
• siRNA sequence making it possible to disrupt the expression of the MARK3 proteins was chosen according to the cDNA sequence of the 2C10 clone (see FIG. 9). Briefly, two sequences are selected for the creation of an siRNA, namely: the sense sequence which has a length of 19 bases homologous to a part of the sequence of
• 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).
• the U373 glioblastoma cells in culture were transfected with the siRNAs whose sequences are shown in FIG. 13. After 5 days of maintenance in a growth medium, the cells are counted and the proliferation rate, calculated on the basis of 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.
• the human glial tumor cell lines (glioblastomas) named U87 and U373.
• SiRNAs are transfected into cultured cells by a conventional method (use of oligofectamine transfection agent, siRNA at 450 nM concentration). Cell survival is measured after transfection.
• the "Middle” condition corresponds to the control without transfection.
• the "GFP” conditions correspond to a transfection with a siRNA directed against the GFP protein (transfection safety controls).
• Conditions 2Cl 0-3 and MARK3 3X correspond to the impact of siRNAs directed against the transcript of the 2C10 antigen and against MARK3.
• the so-called "MARK3" sequence targets a region of the identical transcript between the different MARK3 variants.
• Antisense sequence 5 'ACA-GCA-CUA-UUC-CUG-AUC-A 3' Condition 2C10-3 corresponds to the use of an siRNA that is directed against the original sequence portion of the transcript that encodes the sequence of the 52 amino acids that sign the original variant.
• the siRNA 2Cl 0-3 shows a good cytotoxic activity (as shown in the histogram transmitted last Tuesday), greater than that obtained with the siRNA MARK3.

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